Biophysics.

Researchers in the Biophysics area at BIFI apply experimental and computational tools and methodologies in an interdisciplinary environment for understanding the behavior of biological systems, from molecules (proteins, nucleic acids, small molecules, etc.) to whole organisms and ecosystems, from a quantitative perspective. Proteins and organisms with biotechnological and/or biomedical relevance are of special interest. Research within this area has many applications in different fields:

Biotechnology (protein stabilization and modeling, protein function design, molecular machines, nanostructures, molecular modeling, electronic transport, catalysis, electromagnetic interaction with matter, and protein glycosylation and its role in signaling)

Biomedicine (drug discovery and design, pharmacological target identification and validation, protein-DNA interaction)

Biology (epidemiology, evolution, complex networks)

The Biophysics area at BIFI encompasses 8 lines of research:

  • DeveProtMol: Protein folding and molecular design
  • Biomolecular Interactions
  • Protein glycosylation and its role in disease
  • Complex systems and networks
  • Physical modeling of biomolecules
  • Molecular dynamics and electronic structure
  • Flavoenzymes: action mechanisms and biotechnology
  • Protein Misfolding and Amyloid Aggregation
  • Clinical Diagnosis and Drug Delivery
DeveProtMol: Protein Folding
and Molecular Design

Head of the Research Line:

Javier Sancho

Researchers:

Dr. Olga Varea
Dr. Juan José Galano
María Conde
Sandra Salillas
Alejandro Mahía

 

SUMMARY OF THE RESEARCH LINE

The ProtMol group is devoted to the study and improvementfigure1-angarica-et-al-2016 of protein molecules. Our labs at BIFI and at the Faculty of Sciences are fully equipped for integrative research using techniques of Cellular and Molecular Biology, Biochemistry, Biophysics, and Computation. For the last 25 years we have studied the principles of protein stability, folding and binding, and the relationship between protein dynamics and function. Protein stability is a very important topic from both theoretical and practical perspectives, and we still lack enough quantitative understanding of it. In this respect, we have used a variety of models, such as flavodoxin, to develop biotechnological and computational strategies to assist in protein stabilization using rational principles. Molecular Biophysics is one of our main fields of activity.

We are also quite active in the drug discovery field. In recent years, we have investigated protein conformational diseases in order to both understand their molecular causes and to discover small molecules that can be developed into new drug therapies. In this endeavor, we combine classical Biochemistry and Biophysics with novel HTP screening techniques, Bioinformatics, Synthetic Chemistry and in vivo testing. Our projects include testing in animal models novel antimicrobials effective against the human pathogen Helicobacter pylori, and the discovery and rational improvement of pharmacological chaperones to rescue defective human enzymes (e.g. mutated PAH responsible for Phenylketonuria) and to inhibit the aggregation of a number of human peptides and proteins involved in amyloidogenesis.

figure-2-torreblanca-et-al-2012       figure-7-lamazares-et-al-2015

Rather than in techniques, we are interested in problems and, therefore, we usually combine experimental approaches with computational studies, as needed. We have developed atomistic models of the denatured ensemble (ProtSA), a genome scale Prion predictor (PrionScan), a predictor of locally unstable segments of proteins (ProteinLIPS), and Molecular Dynamics tools for the dentification of SNPs involved in conformational diseases.

Our group is pleased to accept talented and motivated doctoral students or postdocs interested in our work. Just contact us.

 

Relevant publications

1.- Exploring the complete mutational space of the LDL receptor LA5 domain using molecular dynamics: Linking SNPs with disease phenotypes in familial hypercholesterolemia.V. E. Angarica, M. Orozco and J. Sancho. Human Molecular Genetics, 25:1233-1246 (2016).

2.- Rational stabilization of complex proteins: a divide and combine approach. Lamazares, Emilio; Clemente, Isabel; Bueno, Marta Velázquez-Campoy A, Sancho J. Scientific Reports, 5, 9129 (2015).

3.- Predicting stabilizing mutations in proteins using Poisson-Boltzmann based models: study of unfolded state ensemble models and development of a successful binary classifier based on residue interaction energies. Jorge Estrada, Pablo Echenique, Javier Sancho. Phys. Chem. Chem. Phys., 17:31044-31054 (2015).

4.- The FurA regulon in Anabaena sp PCC 7120: in silico prediction and experimental validation of novel target genes. A. Gonzalez, V. E. Angarica; J. Sancho; M.F. Fillat. Nucleic Acids Research, 42:4833-4846 (2014).

5.- Improved flavodoxin inhibitors with potential therapeutic effects against Helicobacter pylori infection. J.J. Galano, M. Alías, R. Pérez, A. Velázquez-Campoy, P. S. Hoffman, J. Sancho. Journal of Medicinal Chemistry. 56-15, pp.6248-6258 (2013).

6.- Discovery of novel inhibitors of amyloid b-peptide 1-42 aggregation. L. C. López, S. Dos-Reis, A. Espargaró, J. A. Carrodeguas, M-L Maddelein, S Ventura, J Sancho. J. Med. Chem.55:9521-9530 (2012).

7.- Contribution of disulfide bonds to stability, folding, and amyloid fibril formation: The PI3-SH3 domain case. R. Graña-Montes, N. S. de Groot, V. Castillo, J. Sancho, A. Velazquez-Campoy, and S. Ventura. Antioxidants and Redox Signaling. 16:1-15 (2012).

8.- ProtSA: A web application for calculating sequence specific protein solvent accessibilities in the unfolded ensemble. J. Estrada, P. Bernadó, M. Blackledge, J. Sancho. BMC Bioinformatics. 10:article104 (2009).

9.- Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria. A.L Pey, M. Ying, N. Cremades, A. Velazquez-Campoy, T. Scherer, B. Thöny, J. Sancho and A. Martinez. Journal of Clinical Investigation. 118:2858-2867 (2008).

10.- The mechanism of LDL release in the endosome: Implications of the stability and Ca++ affinity of the fifth binding module of the LDL receptor. X. Arias-Moreno, A. Velazquez-Campoy, JC Rodríguez, M. Pocoví & J. Sancho. J. Biol. Chem. 283:22670-22679 (2008).

11.- The native-state ensemble of proteins provides clues for folding, misfolding and function. N. Cremades, J. Sancho and E. Freire. Trends in Biochemical Sciences, 31:494-496 (2006).

12.- Do proteins always benefit from a stability increase? Relevant and residual stabilization in a three-state protein by charge optimization. Luis A. Campos, Maria M. Garcia-Mira, Raquel Godoy-Ruiz, Jose M. Sanchez-Ruiz and Javier Sancho. J. Mol.Biol.344: 223-237 (2004).

13.- The tryptophan/histidine interaction in α-helices. J. Fernández-Recio, A. Vázquez, C. Civera, P. Sevilla & J. Sancho. J. Mol. Biol.267: 184-197 (1997).

14.- Closure of a tyrosine/tryptophane aromatic gate leads to a compact fold in apoflavodoxin. C.G. Genzor, A. Perales-Alcón, J. Sancho & A. Romero. Nature Structural Biology, 3:329-332 (1996).

15.- Effect of alanine versus glycine in alpha-helices on protein stability. L. Serrano, J.L. Neira, J. Sancho & A.R. Fersht. Nature, 356:453-455 (1992).

 

Main research projects

1.- PIREPRED: Red Transfronteriza de interpretación del cribado neonatal: de la mutación al paciente. EFA086/15 (ERDF-POCTEFA Interreg VA). 01/09/16-31/08/19. IP: Javier Sancho.

2.- Estabilidad de proteínas: principios básicos de los estados (parcialmente) desplegados y estudios moleculares en enfermedades conformacionales. 01/01/14-31/12/16. BFU2013-47064-P: MINECO. IP: Javier Sancho (A. Velazquez, CoIP).

3.- Comprensión, predicción y validación del fenotipo de las mutaciones patológicas: transformando los resultados básicos en herramientas de diagnóstico. 01/12/14-30/11/16. BIO2014-57314- REDT: MINECO. IP: Javier Sancho.

4.- NEUROMED: Diagnóstico y combate molecular de tres enfermedades neurodegenerativas (Parkinson, fenilcetonuria y amiloidosis TTR. ERDF – SUDOE Interreg IVB. 01/05/14-30/06/15. IP: Javier Sancho.

5.- Desarrollo de procedimientos de estabilización de proteínas con actividad retrotranscriptasa encaminados al diseño de nuevos test de diagnóstico molecular. CERTEST BIOTEC S.L. 01/09/14-31/10/1. IP: Javier Sancho.

6.- Estabilidad conformacional de proteinas: principios generales,analisis estabilidad/funcion del r-LDL y busqueda de nuevas chaperonas farmacológicas. 01/01/11-31/12/13. BFU2010-16297.MICINN. IP: Javier Sancho.

7.- Convenio de colaboración para el desarrollo de inhibidores contra la infección por Helicobacter pylori. GENOMA ESPAÑA. 01/12/08-31/12/13. IP: Javier Sancho.

8.- Principios de estabilidad conformaciónal de proteínas, análisis estructural y energético de conformaciones no nativas e identificación de ligandos bioactivos. BFU2007-61476/BMC MEC. 01/10/07-04/10/10. IP: Javier Sancho.

9.- Análisis de nueva algorítmica para acelerar la simulación de plegamiento de proteínas. PLEBIOTIC S.L. 01/05/09-31/12/09. IP: Javier Sancho.

10.- Principios de estabilidad conformacional y estabilización deproteinas sencillas y moderadamente complejas. BFU2004-01411. MCyT. 13/12/04-12/12/07. IP: Javier Sancho.

 

Collaborators

We mantain active collaborations with many national and international groups. See publications. See also project webs: RedMut, Pirepred.

Biomolecular Interactions

Head of the Research Line:

Adrian Velazquez-Campoy

Researchers:

Rafael Claveria-Gimeno (IISA-IACS)
Jose L. Neira (UMH)

 

SUMMARY OF THE RESEARCH LINE

All biological processes can be described as a sequence of interaction events between biomolecules and conformational changes coupled to those interactions. We work on three main aspects, from both basic and applied perspectives, related to biological molecules:

  • Biophysical characterization of proteins with biotechnological and/or biomedical relevance: conformational landscape, interactions with other biomolecules, conformational changes, phase diagrams, cooperative phenomena…
  • Development of experimental methodologies for the study of biomolecular interactions, cooperative phenomena and functional regulation in biological systems.
  • Development and implementation of high-throughput screening procedures for the identification of bioactive compounds able to modulate the function of pharmacological protein targets.

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The biophysical characterization of a protein provides:

  • Information on the protein conformational landscape. The conformational landscape is the set of conformational states, structurally and energetically distinguishable, that are accessible and significantly populated. The distribution of the protein molecules among these conformational states depends on Gibbs free energy of each state, which in turn is a function of the environmental variables (temperature, pH, ionic strength,…), as well as the interaction with other biomolecules or mutations in the sequence. Because each conformational state may exhibit particular functional properties, the conformation and the function of a given protein are regulated through changes in the environmental variables and biochemical signaling (presence of interacting biomolecules) or mutations. This connection between conformation and function constitutes the basis for the allosteric behavior in proteins: modulation and control of protein conformation (and, therefore, protein function) through interactions with other biomolecules (e.g. ions, cofactors, inhibitors, etc.).

    We apply biophysical tools and methods for studying the conformational landscape of proteins of biotechnological and and/or biomedical interest.

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  • Information on the protein function, regulation and evolution. The physiological function of proteins, and its regulation, always relies on the interaction with other molecules. Therefore, the detailed structural and energetic characterization of its interactions is fundamental for understanding its function and regulation. The interaction with structural metal ions and cofactors is of special interest for protein function regulation. In addition, this information might shed light into evolutive aspects: mutation-induced and/or ligand-induced stabilization of alternative conformational and functional states may give rise to new functions.

    We apply biophysical tools and methods to study binding interactions and its coupling with protein stability and the influence on the conformational landscape.

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  • Information on the molecular and energetic basis of a disease. Very often defective proteins are causative agents for diseases. The mutation-induced and/or ligand-induced stabilization of non-native partially (or completely) unfolded states may lead to improper folding, inactivation and/or early degradation of a given protein, or to the formation of toxic aggregates. Likewise, mutations may distort or impair required interactions for proper function. The biophysical study of its conformational landscape provides information on conformational states that may be relevant regarding its function and regulation, as well as information on the impact of mutations on the interactions required for proper protein function.

    We apply biophysical tools and methods to assess the impact of mutations and ligand binding on the stability and function of pharmacological protein targets.

  • Information for establishing strategies for the identification of bioactive compounds. In both infectious diseases (in which the inactivation of a key protein of the life cycle of the pathogen with an inhibitor is the usual therapeutic strategy) as well as in genetic or metabolic disorders (in which both the inactivation of an increased aberrant protein activity with an inhibitor or the rescue of a defective protein activity with a pharmacological chaperone represents alternative therapeutic strategies) it is necessary to modulate and control the function of a given protein. In order to achieve this goal we must identify low-molecular weight compounds: 1) able to interact with the protein; 2) able to modulate appropriately the protein function; 3) avoiding interactions with unwanted targets minimizing side-effects; and 4) exhibiting an optimal pharmacokinetic (ADMET) profile. The biophysical characterization of a protein target provides relevant information about points 1-3, representing a crucial element for designing, validating and optimizing molecular screening procedures.

    We develop and implement high-throughput screening procedures for identifying bioactive compounds against pharmacological protein targets.

 

figurea-new        figureb-new

  • Information for optimizing bioactive compounds (affinity, selectivity, induced-conformational changes in the target…). The biophysical study of protein interactions provides information on the relevant underlying intermolecular interactions (i.e. hydrogen bonds, van der Waals, electrostatic and hydrophobic interactions). The thermodynamic dissection of a given interaction allows estimating the enthalpic and entropic contributions to the Gibbs free energy of interaction. The partition of the Gibbs energy into its enthalpic and entropic terms (thermodynamic profile or signature of the binding) is fundamental for describing that interaction, because those two energetic terms reflect intermolecular interactions of different nature (regarding strength, specificity, susceptibility to mutations, and ability to redesign and engineering). It is known that ligands of similar affinity interacting with a protein will bind differently and will display different binding properties if they exhibit different thermodynamic signature; in particular, their interaction will be driven by different intermolecular forces, they will show different susceptibility to mutations in the protein target, and they will induce different conformational changes. Therefore, not only the binding affinity, but also the complete thermodynamic signature is relevant for understanding the mode of interaction and as a key decision criterion for the optimization of binding affinity and selectivity.
    We develop and implement procedures for the thermodynamic characterization of binding interactions. A biophysical thermodynamic characterization of lead compound binding provides invaluable information for compound optimization.

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Cooperative phenomena are inherent to the behavior of biomolecules and constitute the molecular and energetic basis for protein regulation and allostery. We may distinguish different cooperativity types or levels in biomolecules, which are intimately interrelated:

  • Intrinsic protein structural stability. The conformational landscape and the intrinsic structural and energetic features of the protein conformation are key determinants of its behavior and function. Many proteins fold spontaneously and adopt a structured well-defined native state. Other proteins may populate non-native partially folded states with biological relevance. Moreover, many proteins do not fold into a well-defined structured conformational state unless they interact with another molecule (intrinsically disordered proteins), which may have implications regarding their function and regulation. Interestingly, conformational landscape and intrinsic structural stability are not conserved among structurally homologous proteins.
    We study the structural stability of proteins, with particular interest on intrinsically (partially) unfolded proteins.

 

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  • Coupling between binding and protein conformational changes. Very often protein interactions are accompanied by conformational changes. This is a reflection of the modulation of the conformational landscape of the protein through the interaction with a given ligand. The extent of the conformational change will depend on many factors, in particular, the intrinsic structural stability of the protein and the specific structural and functional features of the ligand. For example, it has been demonstrated the structural determinants in a ligand for binding affinity do not necessarily coincide with those determinants for allosteric regulation (which is mediated through conformational changes coupled to binding). Also, ligand binding induces protein stabilization giving rise to the distinction between strong or weak coupling between conformational changes and binding, as well as between induced-fit and conformational selection. In fact, induced-fit can be considered as a limit case of conformational selection when a very large Gibbs free energy gap exists between the two conformational states interconnected by ligand binding.
    We study conformational changes coupled to ligand binding in proteins, as well as the interplay between folding and binding in protein function regulation and protein binding specificity.

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  • Binding cooperativity. Homotropic cooperativity (binding cooperativity between molecules of the same ligand binding to several sites in a protein) and heterotropic cooperativity (binding cooperativity between molecules of different ligands binding to one or several sites in a protein) constitute the basis for protein function regulation and allostery. In a broad sense, allostery can be defined as the control and modulation of protein conformation (conformational landscape) through ligand binding. Therefore, any protein can be considered an allosterically regulated protein. The most common example is the pH dependency of the structural stability or the binding affinity of a ligand in a given protein; in this particular case, the binding or dissociation of protons taking place at ionizable functional groups in the protein (or in the ligand) alters the energetics of unfolding or ligand binding. Binding affinity and binding cooperativity are not conserved properties among structurally similar ligands.
    We develop and implement methodologies for characterizing and assessing cooperative (homotropic and heterotropic) binding in proteins and its relation to the biological function.

 

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Relevant publications

1.- Biophysical screening for identifying pharmacological chaperones and inhibitors against conformational and infectious diseases. Velazquez-Campoy, J. Sancho, O. Abian, S. Vega. Current Drug Targets 2016 17:1492-1505.

2.- Repositioning Tolcapone as a potent inhibitor of transthyretin amyloidogenesis and associated cellular toxicity. R. Sant’Anna, P. Gallego, L. Robinson, A. Pereira, Ne. Ferreira, F. Pinheiro, S. Esperante, I. Pallares, O. Huerta, R. Almeida, N. Reixach, R. Insa, A. Velazquez-Campoy, D. Reverter, N. Reig, S. Ventura. Nature Communications 2016 7:10787.

3.- Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases. R.M. Buey, R. Ledesma-Amaro, A. Velazquez-Campoy, M. Balsera, M. Chagoyen, J.M. de Pereda, J.L. Revuelta. Nature Communications 2015 6:8923.

4.- Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c. K. Gonzalez-Arzola, I. Diaz-Moreno, A. Cano-Gonzalez, A. Diaz-Quintana, A. Velazquez-Campoy, B. Moreno-Beltran, A. Lopez-Rivas, M.A. De la Rosa.  Proceedings of the National Academy of Sciences USA 2015 112:9908-9913.

5.- Deconvolution analysis for classifying gastric adenocarcinoma patients based on differential scanning calorimetry serum thermograms. S. Vega, M.A. Garcia-Gonzalez, A. Lanas, A. Velazquez-Campoy, O. Abian. Scientific Reports 2015 5:7988.

6.- Improved flavodoxin inhibitors with potential therapeutic effects against Helicobacter pylori infection. J.J. Galano, M. Alias, R. Perez, A. Velazquez-Campoy, P.S. Hoffman, J. Sancho. Journal of Medicinal Chemistry 2013 56:6248-6258.

7.- Allosteric inhibitors of the NS3 protease from the hepatitis C virus. O. Abian, S. Vega, J. Sancho, A. Velazquez-Campoy. PLoS ONE 2013 8:e69773.

8.- The mechanism of allosteric coupling in choline kinase α1 revealed by a rationally designed inhibitor. M. Sahun-Roncero, B. Rubio-Ruiz, G. Saladino, A. Conejo-Garcia, A. Espinosa, A. Velazquez-Campoy, F.L. Gervasio, A. Entrena, R. Hurtado-Guerrero. Angewandte Chemie International Edition 2013 52:4582-4586.

9.- Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase. S. Chopra, A. Palencia, C. Virus, A. Tripathy, B.R. Temple, A. Velazquez-Campoy, S. Cusack, J.S. Reader. Nature Communications 2013 4:1417.

10.- Kinetics and thermodynamics of chlorpromazine interaction with lipid bilayers: Effect of charge and cholesterol. P.T. Martins, A. Velazquez-Campoy, W.L. Vaz, R.M. Cardoso, J. Valerio, M.J. Moreno. Journal of the American Chemical Society 2012 134:4184-4195.

11.- Conformational stability of hepatitis C virus NS3 protease. O. Abian, S. Vega, J.L. Neira, A. Velazquez-Campoy. Biophysical Journal 2010 99:3811-3820.

12. Discovery of specific flavodoxin inhibitors as potential therapeutic agents against Helicobacter pylori infection. N. Cremades, A. Velazquez-Campoy, M. Martinez-Julvez, J.L. Neira, I. Perez-Dorado, J. Hermoso-Dominguez, P. Jimenez, A. Lanas, P.S. Hoffman, J. Sancho. ACS Chemical Biology 2009 4:928-938.

13.- Thermodynamics of zinc binding to hepatitis C virus NS3 protease: A folding by binding event. O. Abian, J.L. Neira, A. Velazquez-Campoy.  Proteins: Structure, Function and Bioinformatics 2009 77:624-636.

14.- Identification of pharmacological chaperones as new therapeutic agents to treat phenylketonuria. A.L. Pey, M. Ying, N. Cremades, A. Velazquez-Campoy, T. Scherer, B. Thöny, J. Sancho, A. Martinez. Journal of Clinical Investigation 2008 118:2858-2867.

15.- Isothermal titration calorimetry to determine association constants for high-affinity ligands. Velazquez-Campoy, E. Freire. Nature Protocols 2006 1:186-191.

 

Main research projects

1.- New infrastructure for the Laboratorio Avanzado de Cribado e Interacciones Moleculares de Aragon (LACRIMA). Diputación General de Aragón y Ministerio de Economía y Competitividad (Proyecto de Infraestructuras Científico-Tecnológicas UNZA15-EE-3250). Universidad de Zaragoza. 2016-2018. PI: Javier Sancho (Universidad de Zaragoza – BIFI).

2.- Between atom and cell: Integrating molecular biophysics approaches for biology and healthcare (MOBIEU). European Cooperation in Science and Technology (eCOST, COST Action CA15126). ARBRE (Association of Resources for Biophysical Research in Europe). 2016-2020. PI: Patrick England (Institute Pasteur).

3.- Validation of a new, quick, non-invasive diagnostic method in serum for early detection of pancreatic cancer (PANCal). Asociación Española de Gastroenterología – Club Español del Páncreas. Instituto Investigación Sanitaria de Aragón, Hospital Clínico Universitario Lozano Blesa, Hospital Universitario Miguel Servet, Hospital de Donosti, Hospital de Barbastro, Universidad de Zaragoza – Instituto BIFI. 2015. PI: Olga Abián (Instituto de Investigación Sanitaria de Aragón (IIS) – Universidad de Zaragoza – BIFI).

4.- Protein stability: Basic principles of (partially) unfolded states and molecular studies on conformational disorders. Ministerio de Economía y Competitividad (BFU2013-47064-P). Universidad de Zaragoza. 2014-2017. PI: Adrián Velázquez Campoy / Javier Sancho (co-IPs) (Universidad de Zaragoza – BIFI).

5.- NEUROMED – Diagnosis and treatment of three neurodegenerative diseases (Parkinson, Phenylketonuria and TTR amyloidosis). ERDF – SUDOE Interreg IVB (SOE4/P1/E831 – Neuromed). Universidad de Zaragoza, Universidad Autónoma de Barcelona, Instituto de Biologia Molecular e Celular (Porto), Universidade de Coimbra, CNRS, INSERM (Languedoc-Roussillon). 2014-2015. PI: Javier Sancho (Universidad de Zaragoza – BIFI).

6.- Bioavailability of amphiphilic ligands – Drugs and metabolites. Ministerio de Ciencia e Innovación (Proyectos de Movilidad – Acciones Integradas, PRI-AIBPT-2011-1025). Universidad de Zaragoza, Universidad de Coimbra. 2012-2013. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

7.- NS3 protease from hepatitis C virus: Identification of competitive and allosteric inhibitors. Ministerio de Ciencia e Innovación (BFU2010-19451). Universidad de Zaragoza. 2010-2013. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

8.- NS3 protease from hepatitis C virus: Competitive and allosteric inhibitors, and molecular basis of drug resistance. Universidad de Zaragoza (UZ2009-BIO-05). Universidad de Zaragoza. 2010. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

9.- Conformational equilibrium of the NS3 protease from hepatitis C virus: Non-native states and identification of a new type of allosteric inhibitors. Diputación General de Aragón (PI044/09). Universidad de Zaragoza. 2009-2011.

10.- Development of inhibitors of the NS3 protease from hepatitis C virus. Ministerio de Educación y Ciencia (SAF2004-07722). Universidad de Zaragoza. 2004-2007. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI)

 

Collaborators

  • Olga Abian (Instituto Aragones de Ciencias de la Salud and BIFI, Spain)
  • Juan Ausio (University of Victoria, Canada)
  • Rui Brito (Universidade de Coimbra, Portugal)
  • Pierpaolo Bruscolini (Universidad de Zaragoza and BIFI, Spain)
  • Jose A. Carrodeguas (Universidad de Zaragoza and BIFI, Spain)
  • Irene Diaz-Moreno (Instituto de Bioquimica Vegetal y Fotosintesis – CSIC, Spain)
  • Maria Fillat (Universidad de Zaragoza and BIFI, Spain)
  • Marcos R. Fontes (São Paulo State University, Brazil)
  • Ernesto Freire (The Johns Hopkins University, USA)
  • Enrique Garcia-Hernandez (Universidad Nacional Autonoma de Mexico, Mexico)
  • Ruben Martinez-Buey (Universidad de Salamanca, Spain)
  • Milagros Medina (Universidad de Zaragoza and BIFI, Spain)
  • Maria João Moreno (Universidade de Coimbra, Portugal)
  • Arturo Muga (Universidad del Pais Vasco, Spain)
  • Jose A. Navarro (Instituto de Bioquimica Vegetal y Fotosintesis – CSIC, Spain)
  • Julian Pardo (Universidad de Zaragoza and IIS-Aragon, Spain)
  • Santiago Ramon-Maiques (CNIO, Spain)
  • David Reverter (Universidad Autonoma de Barcelona, Spain)
  • Javier Sancho (Universidad de Zaragoza and BIFI, Spain)
  • Jayaraman Sivaraman (National University of Singapore, Singapore)
  • Maria A. Urbaneja (Universidad del Pais Vasco, Spain)
  • Salvador Ventura (Universidad Autonoma de Barcelona, Spain)
Protein glycosylation
and its role in disease

Head of the Research Line:

Ramón Hurtado Guerrero

Researchers:

Ramón Hurtado Guerrero
Matilde de las Rivas
Jorge Castro López

 

SUMMARY OF THE RESEARCH LINE

Our group is interested in the study of glycosyltransferases, glycosylhydrolases and carbohydrate-binding proteins/modules involved in human diseases. We use protein X-ray crystallography as the main tool complemented with enzymology, inhibition studies, etc, in order to study the molecular mechanisms of enzymes that are involved in the synthesis, modification and degradation of glycoconjugates, oligo and polysaccharides (see more relevant publications below).

 

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Figure 1. Crystal structure of POFUT2 in complex with the human TSR1 from thrombospondin 1.

We majorly work in Protein O-glycosylation and glycosyltransferases responsible of this post-translational modification. In particular we are currently working in several glycosyltransferases such as Protein O-fucosyltransferases 1 and 2 (POFUT1 and 2), and the large family of GalNAc-Ts. While POFUT1 and 2 fucosylate folded domains such as EFG and TSR domains (Figure 1), respectively, GalNAc-Ts mainly glycosylate unstructured regions present in a large number of proteins such as mucins (Figure 2).

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Figure 2. Crystal structure of GalNAc-T2 in complex with a glycopeptide. This figure also illustrates the striking dynamic observed in this enzyme, in which compact and extended monomeric structures are present in equilibrium with compact dimers.

Furthermore we are interested in the elucidation of the reaction coordinates and the molecular mechanism by using transition state analogues or Michaelis complexes. Finally these studies will be important for the design of new future drugs with potential therapeutic applications

Although not related to the Glycobiology field, we have a close collaboration with Prof. Guadix and Dr Conejo-García from University of Granada in the development of new inhibitors against human choline kinase α1.

 

Relevant publications

1.- Structural insights into mechanism and specificity of O-GlcNAc transferase. Clarke AJ*, Hurtado-Guerrero R*, Pathak S*, Schüttelkopf AW*, et al. EMBO J. 2008, 27(20): 2780-8. *equal contributation as first author.

2.- Molecular mechanisms of O-GlcNAcylation. Hurtado-Guerrero R, Dorfmueller HC, van Aalten DM. Curr Opin Struct Biol. 2008, 18(5): 551-7.

3.- Recent structural and mechanistic insights into post-translational enzymatic glycosylation. Ramon Hurtado-Guerrero* and Gideon J Davies. Current Opinion in Chemical Biology. 16(5-6):479-87, 2012. *corresponding author

4.- The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. Wong E, Vaaje-Kolstad G, Ghosh A, Ramon Hurtado-Guerrero, et al. PLoS Pathogen, 2012.

5.- The mechanism of allosteric coupling in choline kinase α1 revealed by a rationally designed inhibitor. María Sahún-Roncero, Belén Rubio-Ruiz, Giorgio Saladino, Ana Conejo-García, Antonio Espinosa, Adrián Velázquez-Campoy, Francesco Luigi Gervasio, Antonio Entrena and Ramon Hurtado-Guerrero*. Angewandte Chemie (selected as VIP), 52(17):4582-6, 2013. *corresponding author

6.- Combined structural snapshots and metadynamics reveal a substrate-guided front-face reaction for polypeptide GalNAc-transferase T2. Erandi Lira-Navarrete, Javier Iglesias-Fernández, Wesley F. Zandberg, Ismael Compañón, Yun Kong, Francisco Corzana, B. Mario Pinto, Henrik Clausen, Jesús M. Peregrina, David Vocadlo, Carme Rovira* and Ramon Hurtado-Guerrero*. Angewandte Chemie International Edition, 53(31):8206-10, 2014. *corresponding author

7.- Dynamic interplay between catalytic and lectin domains of GalNAc transferases modulates protein O-glycosylation. Erandi Lira-Navarrete, Matilde de las Rivas, Ismael Compañón, María Carmen Pallarés, Yun Kong, Javier Iglesias-Fernández, Gonçalo J. L. Bernardes, Jesús M. Peregrina, Carme Rovira, Pau Bernadó, Pierpaolo Bruscolini, Henrik Clausen, Anabel Lostao, Francisco Corzana, and Ramon Hurtado-Guerrero*. Nature Communications, 5;6:6937, 2015. *corresponding author

8.- X-ray Structures decipher the Non-equivalence of Serine and Threonine O-glycosylation points: Implications for the Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody. Nuria Martínez-Sáez,‡ Jorge Castro-López,‡ Jessika Valero-González,‡ David Madariaga, Ismael Compañón, Víctor J. Somovilla, Míriam Salvadó, Juan L. Asensio, Jesús Jiménez-Barbero, Alberto Avenoza, Jesús H. Busto, Gonçalo J. L. Bernardes, Jesús M. Peregrina,* Ramón Hurtado-Guerrero,* and Francisco Corzana*. Angewandte Chemie International, 54(34):9830-9834, 2015. *corresponding author

9.- Mucin Architecture behind the Immune Response: Design, Evaluation and Conformational Analysis of an Antitumor Vaccine Derived from an Unnatural MUC1 Fragment. Martínez-Sáez N, Supekar NT, Wolfert MA, Bermejo IA, Hurtado-Guerrero R, Asensio JL, Jiménez-Barbero J, Busto JH, Avenoza A, Boon G-J, Peregrina JM, and Corzana F. Chemical Science, 2016. DOI: 10.1039/C5SC04039F

10.- A proactive role of water molecules in acceptor recognition by Protein-O-fucosyltransferase 2. Jessika Valero-González, Christina Leonhard-Melief , Erandi Lira-Navarrete, Gonzalo Jiménez-Osés, Cristina Hernández-Ruiz, María Carmen Pallarés, Inmaculada Yruela, Deepika Vasudevan, Anabel Lostao, Francisco Corzana, Hideyuki Takeuchi, Robert S. Haltiwanger, and Ramon Hurtado-Guerrero*. Nature Chemical Biology, accepted manuscript, 2016. DOI:10.1038/nchembio.2019. * corresponding author

11.- A trapped covalent intermediate of a glycoside hydrolase on the pathway to transglycosylation. Insights from experiments and QM/MM simulations. Lluís Raich, Vladimir Borodkin, Wenxia Fang, Jorge Castro-López, Daan van Aalten*, Ramon Hurtado-Guerrero* and Carme Rovira*. Journal of the American Chemical Society (JACS), 2016. DOI: 10.1021/jacs.5b10092. * corresponding authors

12.- Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine causing Parasite Death. Lucía Serrán-Aguilera, Helen Denton, Belén Rubio-Ruiz, Borja López-Gutiérrez, Antonio Entrena, Luis Izquierdo, Terry K. Smith*, Ana Conejo-García* and Ramon Hurtado-Guerrero*. Just accepted in Scientific Reports, 2016. *corresponding authors

 

Main research projects

1.- Study of glycosyltransferases involved in the Notch signalling pathway. MICINN, 2011-2014. 120,000 euros. PI: Ramón Hurtado-Guerrero.

2.- Study of protein-carbohydrate interactions involved in human diseases. MEC, 2014-2016. 76,000 euros. PI: Ramón Hurtado-Guerrero.

 

Collaborators

  • Prof. Robert Haltiwanger, The University of Georgia
  • Prof. Henrik Clausen, University of Copenhagen
  • Prof. Daan van Aalten, University of Dundee
  • Prof. Philip Hardwidge, Kansas State University
  • Prof. Tom Gerken, Case Western Reserver University
  • Prof. Pedro Merino, Universidad de Zaragoza
  • Dr. Francisco Corzana, Universidad de La Rioja
  • Dr. Gonzalo Jiménez-Osés, Universidad de La Rioja
  • Prof. Carme Rovira, Universidad de Barcelona
  • Dr. Filipa Marcelo, New University of Lisbon
  • Dr. Julián Pardo, Universidad de Zaragoza
  • Dr. Anabel Lostao, Universidad de Zaragoza
Complex Systems
and Networks (COSNET Lab)

Head of the Research Line:

Professor Yamir Moreno

Researchers:

Yamir Moreno (Group Leader)
Sandro Meloni (Researcher)
Carlos Gracia-Lázaro (Researcher)
Emanuele Cozzo (Postdoctoral researcher)
Sergio Arregui (PhD Student)
Alberto Aleta (PhD Student)
Felipe M. Cardoso (PhD Student)
Pablo Piedrahita (PhD Student)

BIFI members linked to the research line who work at the Department of Condensed Matter Physics, Faculty of Sciences, University of Zaragoza:

Professor Luis Mario Floría Peralta
Jesús Gómez-Gardeñes

 

MAIN LINE OF RESEARCH

The Complex Systems and Networks Lab (COSNET) was founded in 2003 by Professor Yamir Moreno and has an extensive scientific background in Statistical Physics and in the Physics of Complex Systems. During the last decade, the group has consolidated as a leading team at international level in the study of several topics related to the structure and dynamics of Complex Networks, Epidemiology, Systems Biology, Multiplex Networks, synchronization phenomena, technological and social networks, specially, in the emergence and evolution of online social movements, Evolutionary Game Theory, as well as in the analysis of human collective behavior and in the development of large-scale experiments to study cooperation in humans.

Complex Network Theory

Networked systems are all around us. The accumulated evidence that complex systems cannot be fully understood by studying only their isolated constituents, has given rise to the birth of a new movement of interest and research in the study of complex networks. The expectancy is that understanding and modeling the structure of a complex network would lead to a better cottoning on its dynamical and functional behavior. Though modern network theory has produced a number of relevant results in the last few years, it is still in its infancy, particularly, when it comes to applications in real systems and to the comprehension of the relation between structure and function (dynamics). The main purpose of this research line is the study of complex networks and the collective behavior of dynamical agents that interact among them following the couplings given by the topology of these complex networks. As a result, the fundamental objectives are:

  • The simultaneous characterization of the interactions and dynamics at a local scale and the study of their integration into a global and coherent dynamics at a system-wide scale.
  • The study of how global dynamics affects local interactions.
  • The statistical characterization of real networks.
  • The design of realistic models.
  • The study of other dynamical processes on complex networks and the emergence of collective behavior.
  • The development and application of analytical tools to study complex networks.
  • Fostering of a community of multidisciplinary scientists, who master the discipline of complex systems and use it for their research.
  • Identify the best course of action to transfer the acquired knowledge from basic sciences to the application level for a proper characterization and exploitation of real systems.

Complex network theory is particularly advantageous to explore several aspects of complexity. The fundamental idea is to discover the structure of interactions between the components of the system and the emergent behavior of many-body systems coupled to the underlying structure. This would improve our understanding and modeling capabilities so that we may control or predict the dynamics and function of complex networked systems. In addition, this approach does not rely on a detailed knowledge of the system’s constituents, but in the analysis of the relationship between them and therefore, allows obtaining universal results that can be generalized with relative ease (the study of epidemic spreading processes is equivalent to the spread of computer viruses). For example, biological networks like protein interaction networks, share many structural (scale-freeness) and dynamical (functional modules) features with other seemingly different systems such as the Internet and interaction patterns in social systems. Thus, systems as diverse as peer-to-peer networks, neural systems, socio-technical phenomena or complex biological networks can be studied with a unique theoretical and computational tool.

On the other hand, there are still many unknown systems and processes in which the new discoveries and techniques developed in the last years can shed light and provide novel results. For example, why scale-free networks are ubiquitous in Nature? Are there universal principles that govern the growth and evolution of these networks? How the dynamics of local interactions are spread and integrated into the global scale? And, in turn, how the macroscopic behavior of the system modulates interactions at lower scales? Finally, are there common patterns that can be identified not only structurally, but also in the functional organization of these systems? The latter and other questions should be studied by adopting new perspectives and approaches based on multidisciplinary research. The research results have important applications in problems such as: the modeling of biological processes at the molecular and cellular levels (metabolism, gene expression, etc.), the study of epidemic dynamics, the characterization of transport and diffusion processes in networks and communication technologies, several synchronization phenomena and the emergence of collective effects with applications in neuroscience and social systems. The ultimate goal is to understand the general principles governing these biological, social and technological systems in order to be able to predict, design and control the behavior of a wide range of real systems.

“The Science of Complexity”

The Science of Complexity has become extremely important in the study of many real systems. Complexity theory is based on the holistic principle antireductionist that considers the whole greater than the sum of its parts. This perspective allows an accurate analysis of different phenomena so as to predict the evolution of them over time. Complex behavior occurs when many interactions at the local scale collectively lead to unpredictable larger-scale outcomes. However, only very recently scientists have started to reconsider the traditional reductionism viewpoint that has frequently driven science. The accumulated evidence that systems as complex as a group of social animals, or the cells of a living system, cannot be fully understood by simply reducing them to a sum of their fundamental parts, has produced an increasingly large interest in the study of complex systems. These studies are revealing and explaining a range of emergent system behaviors and providing a deeper understanding of entire systems and their responses, with often surprising and unexpected results.

Multiplex Networks

Until very recently, most of the studies on the structure and dynamics of networks considered them as single layer graphs, in which all the interactions, no matter whether they were obtained in different conditions, appeared aggregated. However, as more data about real systems became available, it was evident that many systems are indeed made up by different interaction layers that are interdependent. Think of, for instance, a transportation system, where different transportation modes coexist in space. An accurate representation of such a system would represent each mode of transportation as one layer, with connections among them representing the possibility of commuting from one to another: from a bus line to a metro line, to a tram, etc. The same applies to online social systems, where an individual could be in several platforms (Twitter, Facebook, Google Plus, etc.) concurrently, thus communicating and being exposed to info through many information channels. A final example in the biological domain is given by cellular processes, where there are many biochemical pathways operating at the same time.

This new structural paradigm poses important challenges. First, we need to figure out when the multilayer structure is important and then, develop new metrics, algorithms and representations of such systems. In addition, dynamical processes such as diffusion and spreading dynamics should be reassessed, as it is expected that new phenomenology arises. In our group, we have been studying these networks since a few years and have already produced a significant number of contributions that have had a deep impact on the general subject of network science. This includes the study of diffusion processes, the spreading of multistrain and interacting diseases, new metrics and formalisms to characterize the structure of multilayer networks and the dynamics of multichannel information dissemination. We continue to explore this line and plan to tackle several interesting problems in the biological domain.

multiplexnetworks_researchline_cosnet

 

Epidemiology

Epidemic spreading is a central issue in a variety of fields. In this context, both epidemic modeling and data collection about contact networks are intensively contributing to development of an accurate computational and theoretical framework to simulate how diseases spread and evolve as well as to the search of efficient immunization and vaccination policies. However, the lack of a complete description of connectivity maps and the singularities of the transmission mechanisms make the analysis extremely difficult. Much effort should still be invested in the design of efficient and reliable epidemic spreading models that benefits from data of connectivity patterns given by social maps of contacts (directionality, age, gender, etc.) and also to understand the dynamics of multi-strain or interacting diseases. Our research is mainly focus on the study of situations in which multiple pathogens coexist within the same host population, including systems of competing pathogens (e.g., seasonal influenza) or the so-called syndemic systems (e.g. HIV and Tuberculosis), i.e., the convergence of two or more diseases that act synergistically in a population to magnify the burden of disease. For these latter scenarios, considering multiple networks of contacts or layers is paramount.

tb_disease_modeling

Epidemiological Framework & Multilevel Approach

The study of epidemic spreading processes from a quantitative point of view is a vast field under intense investigation for nearly a century. Mathematical and computational modeling of infectious diseases is a collective endeavor of scientists coming from many scientific fields, from applied mathematicians and epidemiologists, to computer scientists and physicists. Physicist’s approaches to problems in Epidemiology invoke statistical physics, the theory of phase transitions and critical phenomena, to grasp the macroscopic behavior of epidemic outbreaks. It is not adventurous to claim that one of the main successful frameworks is the Mean-Field (MF) approximation, where homogeneity and isotropy are hypothesized to reduce the complexity of the system under study. This approach is mainly used to study the spreading of a disease in a system in which individuals are identified with the nodes of a (complex) network of contacts. Another important alternative approach aimed at describing the large-scale spreading of infectious diseases mathematically is the so-called meta-population approach. This framework describes a set of spatially structured interacting subpopulations as a network whose links denote the mobility of individuals across subpopulations. Each subpopulation consists of a number of individuals that are divided into several classes according to their dynamical state with respect to the modeled disease, for instance: susceptible, infected, removed, etc. The internal compartmental dynamics models the contagion dynamics by considering that people in the same subpopulation are in contact and may change their state according to their interactions and the disease dynamics. Finally, subpopulations also interact and exchange individuals due to mobility from one subpopulation to another.

Being able to understand analytically how the various model components influence the dynamics and interact is not always possible. More often, this is achieved numerically. We therefore pay particular attention to the development of efficient numerical methods that complement, when possible, analytical insights. The constraint of modeling a real process in which myriads of sources of complexity are involved makes the combination of massive data bases with relatively simple models the only sensible approach to model fitting, projection and understanding. Finally, it is worth mentioning that epidemiological research nowadays faces problems related to the lack of appropriate, disease-specific theoretical and computational models to understand the transmission mechanisms behind global public health threats. This is amplified by our current limited knowledge about the interplay among the various scales involved in the transmission of infectious diseases at the global scale.

Most epidemic models are developed assuming that the spreading process takes place on a single level (be it a single population, a meta-population system or a network of contacts). Therefore, pressing problems rooted at the interdependency of multi-scales call for the development of a whole new set of theoretical and simulation approaches. Our research is related the emergence and evolution of global diseases from the new paradigm of multi-scale, interdependent complex systems. Taking into consideration not only a single interaction level but also the interdependency between many levels and scales is a radical change, a change that makes a substantial difference as it allows to address problems such as comorbidity and the spreading of persistent infections and multi-strain diseases, among others. The outcomes may be rewarding and the resulting framework would be invaluable to better comprehend global health threats.

As a result, our goal is to develop a contemporary epidemiological framework that integrates the many aspects involved in the spreading of global diseases: from single networks of contacts to metapopulation systems, to the interaction between different diseases and strains and to the influence of human behavioral changes and mobility patterns.

In this context, our main objectives are:

  • To integrate individual-level approaches into meta-population schemes with the aim of adding 
further realism to the structure of subpopulations.
  • To generate new mathematical and computational modeling schemes to accurately describe the 
impact of human behavioral changes on the course of an epidemic.
  • To develop models that will allow addressing the effects of the interaction between persistent 
infections (and specifically, between TB and AIDS) in structured populations.
  • To significantly advance in a brand new theoretical and modeling framework to better grasp the role 
of competition between cross-reacting strains of infection in multi-scale diseases.
  • To compare the structural and dynamical properties of epidemic multi-scale models looking for similarities, organizational principles and universal dynamical patterns with other multi-level, interdependent complex systems.

disease_reserach_image

Structure and Dynamics of Online Social Systems

The collection and mining of large amounts of digital data is allowing gaining deep insights into the structure and dynamics of large-scale socio-technical systems. In order to advance more in our current understanding of such systems, we need to develop a proper and efficient way to handle these datasets as well as to implement new algorithms and modeling tools that will ultimately allow to extract new knowledge from them. In this respect, it has become apparent that online social systems are playing a role in the development of different collective phenomena that manifest in the off-line world. Therefore, the analysis of the emergence and evolution of influential social movements in social platforms like Twitter is extremely useful if we aim at making further predictions about human collective behavior.

Today, techno-social systems pervade our world. ICT systems are leading to profound transformations in the way our society self-organize and generate and use information as well as on how humans interact, travel, behave, etc. Such transformations are not always traduced in an improved human society, as they can also lead to new forms of instabilities or make social and economic systems more fragile. Moreover, the ongoing technological revolution is taking place at a never-saw pace, which makes it even more urgent the development of tools that allow understanding the emerging properties of complex techno-social systems and to anticipate the consequences of new regulations, actions, or systems’ failures.

Although outstanding results have recently been obtained in modeling collective behavior in techno- social systems, we have not yet progressed enough in basic theoretical aspects and in the application of the generated knowledge to the characterization and understanding of real social phenomena. Moreover, with the unprecedented amount of data at our disposal nowadays, new challenges arise.

Present-day tools simply fail to keep up with the shifting challenges that the interaction between humans and complex ICT systems poses. For instance, think of a techno-social system like online social networks in which individuals engage in a multitude of categorical (politics, science, sports, technology, etc.) layers, giving to each of them a specific weight. Could we predict or simply understand how likely it is that a given rumor, idea or belief reaches system-wide proportions? Is there a general mechanism behind such kind of social phenomenon or are there different mechanisms unique to each categorical level or social niche? Are influential individuals globally defined or instead are they system’s dependent? Can we understand what gives rise to social uproar as recently witnessed? Or even more, are we in a position to answer those questions that we would ideally like to ask?

onlinesocialsystems_cosnet_line

 

Evolutionary Game Theory and The Socio-Technical Man

Evolutionary Game Theory is a branch of mathematics that analyzes the strategic interaction between rational or irrational agents that can be individuals, groups, corporations, etc. The game consists of a set of players and strategies. Each player earns a reward that depends not only on his/her own strategy, but also on the strategy carried out by his/her competitors. This theory is very useful to reveal and predict human behavior and provides a framework and analytical tools for understanding a wide range of phenomena that occur in real life and are linked to decision-making by individuals or groups of individuals who interact with each other (see contributions of John von Neumann, Oskar Morgenstern and John Nash). The Prisoner’s Dilemma is the most studied model in Evolutionary Game Theory and is a symmetric, bi-personal, finite, static and non-zero-sum game in which players must choose between two strategies: Cooperate or defect. From an individual perspective, defect is always the best strategy. Nevertheless, cooperation gives the highest payoff collectively. This is the dilemma.

If we want to describe the Socio-technical man, we need to understand first, some basic problems such as: how humans interact with the environment and other individuals, how cooperative behavior emerges and survives, and how social networks evolve and shape the way we communicate and interact with each other. To this end, we should develop new ways to analyze existing data, perform controlled experiments with groups –of different sizes- of humans facing social dilemmas and hypothetical scenarios and come out with new theoretical and computational methods and algorithms. By doing this, we will be in the position to better understand what are the factors determining human behavior in a plethora of situations, and hopefully, provide hints to, e.g., policy makers, with the aim of creating a better and more sustainable Society for the future. On its turn, given the universality of many concepts and methods of Complexity Science, we are also sure that the new methods will contribute to the development of other areas (for instance, the Physics of non-equilibrium systems, Economics and even Ecology).

Finally, Evolutionary Game Theory is also very useful tool to study different phenomena and biological processes such as the ecology of bacterial population and the evolution of virus and species, in general.

game_theory_research_image

The aim of the Laboratory of Experimental Economics “NECTUNT LAB” is the study of cooperative phenomena in humans as well as the applications of Evolutionary Game Theory in different fields of science. Methodologically, we make use of this theory and computer simulations to build more realistic models based on our findings in controlled experiments, both onsite and online. Currently, we are involved in a European Research Project (IBSEN). The main objective of the project is to develop the largest simulator of human behavior up to date, with the ultimate goal of answering questions such as: What are the mechanisms and real motivations that promote the emergence and evolution of cooperation in humans? How individuals behave in different contexts? How financial bubbles are formed?

On December 20, 2011, the largest experiment developed up to now with thousands of subjects playing a Prisoner’s Dilemma Game took place in Zaragoza, Spain. Almost 1300 students coming from 42 schools and institutes of the Autonomous Community of Aragon were involved in this unprecedented real-time experiment. The design, software and visualization platform were developed by BIFI researchers.

 

Relevant publications

1.- Complex Networks: Structure and Dynamics. S. Boccaletti, V. Latora, Y. Moreno, M. Chávez and D.-U. Hwang. Physics Reports 424, 175-308 (2006).

2.- Heterogeneous networks do not promote cooperation when humans play a Prisoner’s Dilemma. C. Gracia-Lázaro, A. Ferrer, G. Ruíz, A. Tarancón, J. A. Cuesta, A. Sánchez, and Y. Moreno. Proceedings of the National Academy of Sciences USA 109, 12922-12926 (2012).

3.- Evolutionary dynamics of group interactions on structured populations – A review. M. Perc, J. Gómez-Gardeñes, A. Szolnoki, L. M. Floría and Y. Moreno. Journal of the Royal Society Interface 10, 20120997 (2013).

4.- Host mobility drives pathogen competition in spatially structured populations. C. Poletto, S. Meloni, V. Colizza, Y. Moreno and A. Vespignani. PLoS Computational Biology 9 (8): e1003169 (2013).

5.- The role of hidden influentials in the diffusion of online information cascades. R. A Baños, J. Borge-Holthoefer and Y. Moreno. EPJ Data Science 2:6 (2013).

6.- Multilayer Networks. M. Kivela, A. Arenas, M. Barthelemy, J. P. Gleeson, Y. Moreno, and M. A. Porter. Journal of Complex Networks 2, 203-271 (2014).

7.- Behavioral transition with age in social dilemmas: From reciprocal youth to persistent response in adulthood. M. Gutiérrez-Roig, C. Gracia-Lázaro, J. Perelló, Y. Moreno, and A. Sánchez. Nature Communications 5:4362, doi: 10.1038/ncomms5362 (2014).

8.- Dynamics of interacting diseases. J. Sanz, C. -Y. Xia, S. Meloni and Y. Moreno. Physical Review X 4, 041005 (2014).

9.- Reputation drives cooperative behavior and network formation in human groups. J. A. Cuesta, C. Gracia-Lázaro, A. Ferrer, Y. Moreno, and A. Sánchez. Scientific Reports 5:7843 (2015).

10.- Characterizing two-pathogen competition in spatially structured environments. C. Poletto, S. Meloni, A. Van Metre, V. Colizza, Y. Moreno and A. Vespignani. Scientific Reports 5:7895 (2015).

11.- Dynamic instability of cooperation due to diverse activity patterns in evolutionary social dilemmas. C. -Y. Xia, S. Meloni, M. Perc and Y. Moreno, “”, Europhysics Letters 109, 58002 (2015).

12.- Spatiotemporal characterization of information-driven collective phenomena through transfer entropy. J. Borge-Holthoefer, N. Perra, B. Gonçalves, S. Gonzalez-Bailón, A. Arenas, Y. Moreno, and A. Vespignani. Science Advances 2, e1501158 (2016).

13.- Modeling the effects of network structure, competition and memory time on social spreading phenomena. J. P. Gleeson, K. P. O’Sullivan, R. A. Baños, Y. Moreno. Physical Review X 6, 021019 (2016).

14.- Humans conform to a reduced set of behavioral phenotypes when facing social dilemmas. J. Poncela-Casasnovas, M. Gutiérrez-Roig, C. Gracia-Lázaro, J. Vicens, J. Gomez-Gardeñes, J. Perelló, Y. Moreno, J. Duch, and A. Sánchez. Science Advances 2, e1600451 (2016).

 

Main research projects

1.- BRIDGING THE GAP: FROM INDIVIDUAL BEHAVIOR TO THE SOCIO-TECHNICAL MAN (IBSEN), European Commission. H2020 FET Open, Project number 662725, 2015-2018.

2.- DISTRIBUTED GLOBAL FINANCIAL SYSTEMS FOR SOCIETY (DOLFINS), European Commission. H2020 FET Proactive GSS, Project number 640772, 2015-2017.

3.- FOUNDATIONAL RESEARCH ON MULTILEVEL COMPLEX NETWORKS AND SYSTEMS (MULTIPLEX), European Commission. FET Proactive IP Project number 317532, 2012-2016.

4.- MATHEMATICAL FRAMEWORK FOR MULTIPLEX NETWORKS (PLEXMATH), European Commission. FET Proactive STREP Project number 317614, 2012-2015.

 

Collaborators

  • Alessandro Vespignani (Sternberg Distinguished University Professor College of Computer and Information Science, College of Science, Bouvé College of Health Sciences, Northeastern University, Boston, US)
  • Vittoria Colizza (Inserm and UPMC Université Paris 06, Faculté de Médecine, Paris & ISI Foundation, Turin, Italy)
  • Chiara Poletto (Researcher (Chargé de Recherche 2ème classe) EPICX-Lab, iPLESP, INSERM & UPMC UMR-S 1136)
  • Carlos Martín (Group of Mycobacterial Genetics, Faculty of Medicine, University of Zaragoza, Spain)
  • James Gleeson (Department of Mathematics and Statistics, University of Limerick, Ireland)
  • Javier Borge-Holthoefer (Internet Interdisciplinary Institute, IN3, Group: CoSIN3, Barcelona, Catalunya)-External BIFI Member.
  • Sandra González-Bailón (University of Pennsylvania’s Annenberg School for Communication, US)
  • Francisco Rodriguez (Department of Applied Mathematics and Statistics, Institute of Mathematics and Computer Science, University of São Paulo, Brazil)
  • Angel Sánchez (Universidad Carlos III, Madrid, Spain. External BIFI Member)
  • José Cuesta (Universidad Carlos III, Madrid, Spain. External BIFI Member)
  • Josep Perelló (OpenSystems-UB, Departament de Física Fonamental, Universitat de Barcelona)
  • Matjaz Perc (Faculty of Natural Sciences and Mathematics, University of Maribor, Slovenia)
  • Zhen Wang (Research Associate, School of Computer Science and Engineering, Nanyang Technological University)

 

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Physical modeling of biomolecules

Head of the Research Line:

Pierpaolo Bruscolini

Researchers:

Pierpaolo Bruscolini, Researcher UZ
Antonio Rey Gayo, Full Professor UCM
Ana Ma Rubio Caparrós, Associate professor UCM
Fernando Falo Forniés, Associate professor UZ

 

SUMMARY OF THE RESEARCH LINE

The research line of “Physical modeling of biomolecules” is articulated around three research groups which share the same approach to the physics of biological molecules as well as the analysis techniques from statistical mechanics.

Bruscolini’s Group (Statistical-physics modeling of biomolecules)

We apply coarse-grained models and statistical mechanics methods to protein folding, protein function, protein design and sequencing, seeking the best balance between the quantitative accuracy of the predictions (increasing with the complexity of the model) and the viability in terms of computational costs and time (increasing with the simplicity of the approach). We are also interested in statistical inference and its applications, and we are using it to investigate protein signaling and redesign. Our view is that the big-data explosion that we are witnessing in biology can be best tackled with simple models and approaches that help rationalizing and understanding the biological processes.

In more detail, our recent activity encompasses:

a) Protein Folding: we use simple models, and especially the Wako-Saito-Muñoz-Eaton model, for which an exact solution can be given (PRL 88, 258101, 2002). In particular, we have developed the latter model to provide a quantitative description of the experimental data from different experimental techniques.

b) Protein design: we deal with protein design as an optimization process, at fixed backbone, over the choice of the amino acids and their rotamers to be arranged in the protein sequence. Our results suggests that it is important to account for the entropy to design optimal sequences. We are interested in developing the method further, using different force fields for the energy of the rotamers.

c) Protein Sequencing: we are interested in de-novo sequencing by Tandem Mass Spectrometry, and have proposed a novel algorithm, T-NovoMS, which relies on the mapping of the sequencing of a parent peptide on the thermodynamics of a one-dimensional system, with the experimental MSMS spectrum acting as an external field. We have developed a web server implementing the method: http://webapps.bifi.es/tnovoms/

d) Protein function: we have applied a simple worm-like-chain model to explain the interplay between the catalytic and lectine domain of GalNAC transferase 2 in determining the structure and function of the enzyme.

1
Rey’s Group (Computer modeling of protein folding and aggregation)

We use molecular modeling and coarse-grained models to explore the stability of the native structure in proteins and the protein folding process.

We design ourselves the simulation models in order to properly analyze structural, thermodynamic and kinetic aspects of the folding process. The most important part in this design is the interaction potential, which has to permit to reach the native conformation from the unfolded state. In the last decade, we have been mainly devoted to structure-based (Gō-type) models, also known as native-centric models. They consider the contacts between amino acid pairs which can be found in the native structure, in order to define the attractive interactions which rank the different conformations sampled along the simulation. We have designed simulation models which combine these potentials with mean field interactions which take into account the chemical sequence of the considered protein sequence. In addition, we have introduced new interactions to describe the hydrogen bonds present in the protein backbone, which play a fundamental role in the study of the aggregation processes which frequently appear when proteins are kept at moderate or high concentrations.

Our main sampling technique to study the thermal transition between the native and unfolded states of the desired proteins has been an in-house designed Monte Carlo method, together with replica exchange (parallel tempering). Our research work involves a strong methodological part, by dissecting the different physicochemical contributions to the behavior of the simulation models. By using this approach to the problem, we have been able to build models which can be widely used is different systems of interest in relevant scientific and applied problems: two state proteins vs downhill folding, proteins with thermodynamic intermediates, knotted proteins, unfolding transitions under pressure, proteins in highly confined environments or the competition between folding and aggregation.

     2       3

 

Falo´s Group (Modeling and Simulation of biological systems)

We use mesoscopic models at different scales to address different biological problems:

a) Transport by molecular motors. We investigate the transport of cargos inside cell by processive directional motors (kinesin and dynein). Our work is focused now on the study of the influence of microtubule network structure in cargo transport and as a mean of effective motor-motor interaction.

b) Translocation of polymers. The polymers passage through membranes is an important phenomenon both from the point of view of biological and technological processes. We are developing models of translocation driven by time-dependent forces (deterministic or stochastic) to simulate a molecular motor.

c) Free energy landscape of biomolecules: we apply Markov Network Models to the process of mechanical and thermal unfolding of proteins. Besides, we are studying the modelling of the mechanical unbinding of molecular complexes in order to understand the underlying free energy profile.

d) Models of non-canonical DNA structures: We focus on the behavior of guanine quadruplex of DNA (G-quaduplex) under external forces as those generated by AFM or optical tweezers. We are developing mesoscopic models of such molecules as well as performing “all atom” molecular dynamics simulations. Our goal is to bridge the gap between the theory and simulation regimes and the realistic experimental conditions.

e) Other fields of study comprise biological water behaviour, system biology of cellular differentiation, DNA models of denaturation and unzipping.

4

Relevant publications

1.- Steric confinement and enhanced local flexibility assist knotting in simple models of protein folding. Miguel Soler, Antonio Rey, and Patrícia FN Faísca. Phys. Chem. Chem. Phys. DOI: 10.1039/c6cp05086g (2016).

2.- Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Lira-Navarrete, E.; De Las Rivas; Compañón, I.; Pallarés, M. C.; Kong, Y.; Iglesias-Fernández, J.; Bernardes, G. J. L.; Peregrina, J. M.; Rovira, C.; Bernadó, P.; Bruscolini, P.; Clausen, H.; Lostao, A.; Corzana, F.; Hurtado-Guerrero, R. Nature Communications 6: 6937, 2015.

3.- Mapping the Topography of a Protein Energy Landscape. Hutton, R. D.; Wilkinson, J.; Faccin, M.; Sivertsson, E. M.; Pelizzola, A.; Lowe, A. R.; Bruscolini, P.; Itzhaki, L. S. J. Am. Chem Soc. 137 – 46, pp. 14610 – 14625. 2015.

4.- Active polymer translocation in the 3d domain. A. Fiasconaro, J. J. Mazo, F. Falo. Physical Review E 91, 022113 (2015).

5.- An integrative approach for modeling and simulation of Heterocyst pattern formation in Cyanobacteria filaments. A. Torres-Sánchez, J. Gómez-Gardeñes, F. Falo. PLoS Comput Biol 11(3) e1004129. (2015).

6.- Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes. A. E. Bergues-Pupo, J. R. Arias-Gonzalez, M. C. Morón, A. Fiasconaro and F. Falo. Nucleic Acids Research 43(15) 7638-7647 (2015).

7.- How determinant is N-terminal to C-terminal coupling for protein folding?. Heinrich Krobath, Antonio Rey, and Patrícia FN Faísca. Phys. Chem. Chem. Phys. 17, 3512 – 3524 (2015).

8.- Intermediates in the folding equilibrium of repeat proteins from the TPR family. Vicente González and Antonio Rey. Eur. Biophys. J. 43, 433 – 443 (2014).

9.- Design of a rotamer library for coarse-grained models in protein folding simulations. María Larriva and Antonio Rey. J. Chem. Inform. Model. 54, 302 – 313 (2014).

10.- Binary interactions between dendrimer molecules. A simulation study. Ana M. Rubio, Carl C. McBride and Juan J. Freire. Macromolecules, 47, 5379 – 5387 (2014).

11.- MS/MS spectra interpretation as a statistical-mechanics problem. Faccin, M.; Bruscolini, P. Analytical Chemistry. 85 – 10, pp. 4884 – 4892. 2013.

12.- Quantitative prediction of protein folding behaviors from a simple statistical model. Bruscolini, P.; Naganathan, A. N. J. Am. Chem. Soc. 133 – 14, pp. 5372 – 5379. 2011.

13.- Influence of direct motor-motor interaction in models for cargo transport by a single team of motors. S. Bouzat, F. Falo. Physical Biology 7, 046009 (2010).

14.- Computational Protein Design with Side-Chain Conformational Entropy. Sciretti, D.; Bruscolini,P.; Pelizzola,A.; Pretti,M.; Jaramillo,A. Proteins. Structure Function and Bioinformatics 74 – 1, pp. 176 – 191. 2009.

15.- Exploring the free energy landscape: From dynamics to networks and back.
D. Prada-Gracia, J. Gómez-Gardeñes, P. Echenique, F. Falo. PLoS Computational Biology 5(6): e1000415. (2009) 9 pages.

 

Main research projects

1.- FIS2014-55867-P: SocioBioTec: “FÍSICA ESTADÍSTICA Y NO LINEAL APLICADA A SISTEMAS SOCIALES, BIOLÓGICOS Y TECNOLÓGICOS.”
National Project. PI: Juan José Mazo Torres. Funding agency: MINECO. MINISTERIO DE ECONOMIA Y COMPETITIVIDAD. Start-end date: 01/01/2015 – 31/12/2017.

2.- FIS2011-25167: Redes, Biofísica y Ciencia No Lineal. National Project. PI: Juan José Mazo Torres. Funding agency: Ministerio de Ciencia e Innovación. Start-end date: 1-1-2012 / 31-7-2015.

3.- FIS2009-13364-C02-01. “ACERCAMIENTO COMPUTACIONAL A LA COMPLEJIDAD EN REDES,PROTEINAS, Y SISTEMAS DE MUCHOS AGENTES”. Ámbito geográfico: Nacional. PI: Pierpaolo Bruscolini. Funding agency: MINISTERIO DE CIENCIA E INNOVACION. Start-end date: 01/01/2010 – 31/12/2012.

4.- (FIS2008-01240).Dinámica y Estructura de Sistemas Complejos. Ámbito geográfico: Nacional. PI: Juan José Mazo Torres. Funding agency: MINISTERIO DE CIENCIA E INNOVACION. Start-end date: 2009-2011.

5.- FIS2006-12781-C02-01 “COMPLEJIDAD EN PROTEÍNAS, REDES Y SISTEMAS DE MUCHOS AGENTES”. Ámbito geográfico: Nacional. PI: Pierpaolo Bruscolini. Funding agency: MINISTERIO DE EDUCACION Y CIENCIA. Start-end date: 01/10/2006 – 30/09/2009.

6.- FIS2009-13364-C02-02 “Modelos físicos para la simulación de tránsitos conformacionales en proteínas”. National Project. PI: Antonio Rey Gayo. Funding agency:Ministerio de Ciencia e Innovación. Proyecto Start-end date: 2010- 2013.

7.- S2009/PPQ-1551: “Química a alta presión, QUIMAPRES”. Regional Project. PI: Antonio Rey Gayo (at the UCM-SIMPOL group). Valentín García Baonza (coordinator). Funding agency: Comunidad de Madrid. Start-end date: 2010 – 2013.

 

Collaborators

Bruscolini’s group:

  • Alessandro Pelizzola, Dept of Physics, Politecnico di Torino (Italy).
  • Antonio Rey, Dept. of Chemistry, UCM, Madrid (Spain).
  • Laura Itzhaki, Dept. of Pharmacy, Cambridge University (UK).
  • Javier Sancho Sanz, BIFI, Universidad de Zaragoza (Spain).
  • Ramón Hurtado, BIFI, Universidad de Zaragoza (Spain).
  • Milagros Medina, BIFI, Universidad de Zaragoza (Spain).
  • Fernando Falo Fornies, BIFI, Universidad de Zaragoza (Spain).
  • Sergio Perez Gaviro, BIFI, Centro Universitario de la Defensa, Zaragoza, Spain.

Rey’s Group

  • Patrícia F.N. Faísca (University of Lisbon, Portugal).
  • Valentín García Baonza (UCM, Dept. Química Física I).
  • Pierpaolo Bruscolini (UZ y BIFI).
  • Manuel Ángel Ramos (UCM, Dept. Matemática Aplicada).

Falo’s Group

  • Sebastián Bouzat, CEA Bariloche, Argentina).
  • Juan José Mazo (ICMA and Universidad de Zaragoza).
  • Jesús Gómez-Gardeñes (BIFI and University of Zaragoza).
  • Alessandro Fiasconaro (ICMA and University of Zaragoza).
  • María del Carmen Morón (ICMA and University of Zaragoza).
  • Jesús Bergues (Universidad San Jorge, Zaragoza).
  • Pierpaolo Bruscolini (UZ y BIFI).
  • Ricardo Arías González. Instituto de Nanociencia de Madrid (IMDEA).
  • Anabel Lostao. Instituto de Nanociencia de Aragón.
Molecular dynamics
and electronic structure

Head of the Research Line:

Jesús Clemente-Gallardo

Researchers: Permanent members

José Luis Alonso Buj (Departamento de Física Teórica, Facultad de Ciencias)
Alberto Castro Barrigón (ARAID, Edificio I+D)
Jesús Clemente Gallardo (Departamento de Física Teórica, Edificio I+D)
Fernando Falceto Blecua (Departamento de Física Teórica, Facultad de Ciencias)
Víctor Gopar (Departamento de Física Teórica, Facultad de Ciencias)
Víctor Polo Ortiz (Departamento de Química Física, Facultad de Ciencias)

Researchers: PhD students

Adrián Gómez Pueyo (Departamento de Física Teórica, Edificio I+D)
Jorge Alberto Jover Galtier (Departamento de Física Teórica, Facultad de Ciencias)

 

SUMMARY OF THE RESEARCH LINE

In our group, we are mainly concerned with the application of theoretical and computational tools to the study of the behavior of biological and solid state systems. Most of our methods are based on quantum mechanics and in the tools required to combine it efficiently with classical mechanical methods. We deal with many different aspects, from the most theoretical to the most applied. The following are our main lines of work:

A.- Hybrid quantum-classical models: non-adiabatic dynamics of molecular systems

Quantum Mechanics admits a description in terms of tensorial objects defined on the space of physical states. It has been proved very helpful to describe important magnitudes such as the entanglement or the purity of a given system, while being formally analogous to the tensorial description of a classical mechanical system. Both formalisms admit Hamiltonian descriptions for the most common dynamical situations, and this fact allows a straightforward generalization to statistical systems with that type of dynamics for the microstates.

When describing molecular systems, it is common to approximate some degrees of freedom (usually those corresponding to the nuclei) and describe them as classical objects, but there are some degrees of freedom (usually part or the electronic system) which must be described as quantum objects. The tensorial description allows us to combine them in a hybrid quantum-classical description which can be made to maintain several mathematical properties and this provides us with a mathematically rigorous description of the corresponding statistical system. With these tools a firm theoretical background can be constructed to enlarge traditional adiabatic methods such as those based on the Ehrenfest formalism in order to include properties such as decoherence of the electronic dynamics.

Nonetheless, several properties of the resulting models are not fully understood yet. We are studying the main properties of the equilibrium distributions of hybrid systems, in particular in the thermodynamic limit. We are also studying their dynamical properties, in particular what concerns the decoherence of the electronic states. And finally, we are trying to understand ab initio how to model the interaction of a hybrid system with an environment by incorporating stochastic effects in the hybrid dynamics.

B.- Foundations and applications of time-dependent density-functional theory

Density-functional methods have become the most successful techniques for electronic structure calculations; the time-dependent variant is the choice for spectroscopic studies that involve excited electronic states. We develop algorithms and code for the application of these theoretical tools and we belong to the team of developers of the code Octopus (http://www.tddft.org/programs/octopus/)

From the code we also consider relevant applications in Physics, mostly centered on molecules and nanostructures, with focus on the irradiation of the systems with large intensity lasers, and the non-linear phenomena triggered by these usually ultra-short pulses.

C.-Analysis of chemical phenomena using DFT

In this research line, theoretical studies at the DFT level are carried out for molecular systems of interest in the fields of material science and catalysis. Working together with experimental groups of inorganic and physical chemistry, theoretical calculations are nowadays a fundamental tool for the understanding of chemical concepts. Hence, important questions regarding reaction mechanisms, spectroscopic properties or molecular orbital analysis are elucidated from first principles. The theoretical results not only provide a detailed explanation of the obtained results but serve as a guide for the rational design of new molecular species with desired properties.

D.- Quantum optimal control theory

The properties and the microscopic structure of matter can be modified at the electronic level with external knobs: most notably with ultra-fast custom-shaped intense laser pulses, but also through the modification of variables such as temperature, pressure, doping, presence of external static electric or magnetic fields, etc. We propose the use of first-principles electronic structure techniques (for example, time-dependent density-functional theory, TDDFT) in connection with control theories, to design materials, or prepare states of matter, with optimal values of selected properties. The rapidly evolving capabilities of advanced laser sources (pulse durations, intensities, shaping freedom) has triggered intense research in quantum optimal control theory and other control schemes – yet few studies have directly addressed the fast, attosecond scale movement of electrons with an ab initio technique such as TDDFT, that also promises the capacity of dealing with large systems. TDDFT offers the possibility of dealing with highly non-linear response of atomic and molecular systems, even though its accuracy and reliability are permanently improving, and still require theoretical and methodological advances.

E.- Wave transport phenomena in complex systems.

Transport of quantum and classical waves through complex systems has attracted a lot of interest from both fundamental and practical interests. For instance, transport of quantum waves, or matter waves, such as electrons and photons are at the forefront of research in condensed matter. This has been motivated by the creation of new materials such as graphene and topological insulators.

In this research line we investigate the effects of the presence of disorder such as impurities on different quantities related to the quantum transport of electrons through small samples made of graphene, topological insulator materials, and normal metals. The presence of disorder gives a random character to the transport electronic properties of the materials. Thus, in this research line we are particularly interested in studying the statistical properties of quantities, such as the conductance, shot noise, concurrence, etc.

Regarding classical waves, it turns out that many phenomena that one can observe in quantum waves (electrons) can be also seen in classical (macroscopic) systems such as microwave waveguides. This is a signature of the universality of the wave phenomena that we study. Therefore, our theoretical framework has been applied to studied the transmission of electromagnetic waves, and other quantities, in experimental microwave setups.

 

Relevant publications

1. Conductance of 1D quantum wires with anomalous electron-wavefunction localization. Ilias Amanatidis, Ioannis Kleftogiannis, Fernando Falceto, Victor A. Gopar. Phys. Rev. B 85, 235450 (2012).

2. Ehrenfest dynamics is purity non-preserving: A necessary ingredient for decoherence. J. L. Alonso, J. Clemente-Gallardo,J. C. Cuchí, P. Echenique, F. Falceto. Journal of Chemical Physics 137, 054106, 2012.

3. Non-adiabatic effects within a single thermally averaged potential energy surface: Thermal expansion and reaction rates of small molecules. J. L. Alonso, A. Castro, J. Clemente-Gallardo, P. Echenique, J. J. Mazo, V. Polo, A. Rubio and D. Zueco. Journal of Chemical Physics 137, 22A533, 2012.

4. Conductance through disordered graphene nanoribbons: Standard and anomalous electron localization. Ioannis Kleftogiannis, Ilias Amanatidis, Victor A. Gopar. Phys. Rev. B 88, 205414 (2013).

5. Comment on “Correlated electron-nuclear dynamics: Exact factorization of the molecular wavefunction” [J. Chem. Phys. 137, 22A530 (2012)]. J. L. Alonso J. Clemente-Gallardo P. Echenique-Robba and J. A. Jover-Galtier. J. Chem. Phys. 139, 087101, 2013.

6. Beyond Anderson Localization in 1D: Anomalous Localization of Microwaves in Random Waveguides. A. A. Fernandez-Marin, J. A. Mendez-Bermudez, J. Carbonell, F. Cervera, J. Sanchez-Dehesa, and Victor A. Gopar. Phys. Rev. Lett., 113, 233901 (2014).

7. Optimal control of high-harmonic generation by intense few-cycle pulses. Solanpaa, J.; Budagosky, J. A.; Shvetsov-Shilovski, N. I.; et al. Physical Review A 90, 053402. 2014.

8. Nonextensive thermodynamic functions in the Schrödinger-Gibbs ensemble. J. L. Alonso, A. Castro, J. Clemente-Gallardo, J. C. Cuchí, P. Echenique-Robba, J. G. Esteve and F. Falceto. Phys. Rev. E 91 022137, 2015 .

9. NH Activation of Ammonia by [{M(-OMe)(cod)}2] (M = Ir, Rh) Complexes: A DFT Study. Vélez, E.; Betoré M. P.; Casado, M. A.; Polo, V. Organometallics, 2015, 34, 3959–3966.

10. Solvent-Free Iridium-Catalyzed Reactivity of CO2 with Secondary Amines and Hydrosilanes. Julian, A.; Polo, V.; Jaseer, E. A.; Fernandez-Alvarez, F. J.; Oro, L. A. ChemCatChem 2015, 7, 3895–3902.

11. Enhancing and controlling single-atom high-harmonic generation spectra: a time-dependent density-functional scheme. A. Castro, A. Rubio, and E. K. U. Gross. Eur. Phys. J. B 88, 191 (2015).

12. Oxidative Addition of the N–H Bond of Ammonia to Iridium Bis(phosphane) Complexes: A Combined Experimental and Theoretical Study. Betoré, M. P.; Casado, M. A.; García-Orduña, P.; Lahoz, F. J.; Polo, V.; Oro, L. A. Organometallic, 2016, 35, 720-731.

13. Alkoxycarbonylation of α,β-Unsaturated Amides Catalyzed by Palladium (II) Complexes: A DFT Study of the Mechanism. Suleiman, R. K..; Polo, V.; El Ali, B. RSC Advances, 2016, 6, 8440-8448.

14. Theoretical shaping of femtosecond laser pulses for molecular photo-dissociation with control techniques based on Ehrenfest’s dynamics and time-dependent density-functional theory. Alberto Castro. ChemPhysChem 17, 1439 (2016).

15. Tailored pump-probe transient spectroscopy with time-dependent density-functional theory: controlling absorption spectra. Jessica Walkenhorst, Umberto De Giovannini,1, Alberto Castro, and Angel Rubio. Eur. Phys. J. B 89, 128 (2016).

 

Main research projects

1.- FP7-NMP-2011-SMALL-5: Dynamics and control in nanostructures for magnetic recording and energy applications, 7th framework programme, EU

2.- FIS2013-46159-C3-2-P. TEORÍA DE SISTEMAS HÍBRIDOS CLÁSICO-CUÁNTICOS: EQUILIBRIO, DINÁMICA Y CONTROL., 01/07/2014-31/12/2017, 45980€, IP: Alberto Castro

3.- FIS2014-61301-EXP: “Una ruta nueva en la bsqueda del funcional exacto de la teorı́a de funcionales de la densidad” (. Entidad financiadora: Ministerio de Economiı́a y Competitividad. Investigador principal: Alberto Castro. Financiación: 35000€ Duración del proyecto: 09/2015 – 08/2017

4.- CTQ2012-35665 DISEÑO DE CATALIZADORES ORGANOMETÁLICOS PARA LA ACTIVACIÓN Y FUNCIONALIZACIÓN DE AMONIACO: ESTUDIOS COMPUTACIONALES Y EXPERIMENTALES., IP. Víctor Polo, (2013-2015), 80730€

5.- CTQ2015-67366-P: ACTIVACIÓN DE NH3 Y CO2 POR COMPLEJOS DE RODIO E IRIDIO Y SU APLICACIÓN EN EL DESARROLLO DE PROCESOS CATALÍTICOS PARA LA SÍNTESIS DE COMPUESTOS DE ALTO VALOR AÑADIDO, IP Víctor Polo – Miguel Angel Casado (2016-2018), 112651€

 

Collaborators

  • Juan Carlos Cuchí, Universidad de Lérida, Spain
  • Ángel Rubio, Universidad del Pais Vasco, Spain
Flavoenzymes: action mechanisms
and biotechnology

Head of the Research Line:

Dr. Milagros Medina

Researchers:

Dr. Marta Martínez Júlvez
Dr. Patricia Ferreira Neila
Dr. Raquel Villanueva Llop
Dr. Guillermina Goñi Rasia
María Sebastián Valverde
Silvia Romero Tamayo
Ernesto Anoz Carbonel

 

SUMMARY OF THE RESEARCH LINE

Flavoenzymes carry out multiple functions in all type of organisms due to their great versatility, the best known being those involved in electron transfer, oxido-reduction and dehydrogenation processes. Moreover, some have also been identified as redox switches for the control and the modulation of cellular processes mediated by other biomolecules. In this context, new horizons open for the understanding of their action mechanisms and of the flavin-dependent lifestyles, as well as in the development of biotechnological strategies based on these enzymes. Flavoenzymes are common components of key metabolic processes that rely on oxido-reduction reactions due to their versatility and unique ability to connect processes of two electrons with those of a single one. They can participate in redox processes because the isoalloxazine moiety of their FMN or FAD cofactors is a redox agent that can exist in three different states: fully oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (hydroquinone). The large versatility of FMN and FAD in vivo can only be understood when the flavoprotein is considered as a whole, since they only act as successful cofactors when their reactive potentialities are tuned by the protein. This makes each flavoprotein highly specific with respect to electron partners and reactions. Therefore, flavoenzymes can be considered like efficient and sophisticated molecular instruments that use molecular recognition to control redox processes. Therefore, apart from their physiological functions they can be key instruments in biomedicine and biotechnology, acting as drug targets, in the development of antimicrobials or in the control of human diseases, and providing essential chemicals for different human activities. Despite this, the biotechnological use of these enzymes is still limited. This is a consequence of our pour understanding that prevents us to control of all the factors that govern their behaviors, stabilities and, particularly, their catalytic activities. Our hypothesis is that we need to understand the molecular mechanisms behind key metabolic flavoenzymes from bacteria, parasites, plants or animals, to exploit their potentials in different areas of the biotechnology. In our group, we apply an interdisciplinary combination of biochemical, biophysical, cell biology and computational tools to investigate the mechanism, as well as the factors that provide versatility, of several systems depending on flavoenzymes to use such information in new biotechnological and therapeutic applications. Publications from the group reflect contributions from across these key discipline areas of flavoenzymes. Given the wide interest and methodologies in our research, we have strong collaborations with specialists in other disciplines, within BIFI and UNIZAR, as well as with those external to our Institution.

 

Biosynthesis of flavin cofactors: Prokaryotic bifunctional enzymes as drug targets.

fig-2-mmedinaRiboflavin (RF) can be de novo synthesized by plants, yeast and most prokaryotes, but RF uptake from the environment is essential for human nutrition and animal feeding. All organisms are able to transform RF, first into FMN, and then into FAD, by the sequential action of two activities, an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, whereas eukaryotes and archaea use two different enzymes for FMN and FAD production, most prokar yotes depend on a single bifunctional enzyme, FAD synthetase (FADS). Deficiency of RFK or FMNAT activities prevents assembly of flavoproteins essential for cell survival. Moreover, changes in the RFK and FMNAT expression levels cause several cellular stresses, suggesting they are also involved in diverse cellular functions.

Differential molecular characteristics to carry out the same chemistry between the enzymatic components for FMN and FAD production in prokaryotes and eukaryotes have been revealed, making selective inhibition of prokaryotic FADS a feasible treatment for diseases produced by pathogens, and, therefore, worth to be explored. The first stage in this process is to increase the structural-functional knowledge of all these enzymes, but particularly prokaryotic FADS. In this line, using as model the FADS from Corynebacterium ammoniagenes we have determined the binding and catalytic parameters for the substrates at the two catalytic sites, and localized them in each one of the modules of this enzyme. No homology is found among the FADS module responsible for the FMNAT activity and the monofunctional FMNAT enzymes in mammals. Moreover, despite the homology at the RFK module of FADS with regard to the corresponding monofunctional enzymes in eukarya, a much more complex structural reorganization is envisaged for the prokaryotic protein. Finally, the quaternary organization identified for FADS from Corynebacterium ammoniagenes suggests a possible role of this protein in the flavin homeostasis in prokaryotes. Studies are also under progress with FADS from human pathogens, such as Streptococcus pneumoniae or Listeria monocytogenes.

figure-3-mmedina    figure-4-mmedina

The human apoptosis inducing factor (AIF)

Multicellular organisms have developed complex mechanisms to eliminate potentially dangerous body cells by a mechanism of programmed death cell called apoptosis. Deregulation of this mechanism is involved in a number of carcinogenic processes. A cysteine proteases family (caspases) was identified as responsible for apoptosis, but the existence of an alternative mechanism involving the apoptosis inducing factor (AIF) was evidenced. AIF is a phylogenetically preserved mitochondrial flavoenzyme sharing homology with different families of reductases. In intact mitochondria, AIF presents a NADH-oxido-reductase activity through its flavin cofactor, FAD, that has been linked to the stability and biogenesis of the oxidative phosphorylation complexes I and III through the interaction with other proteins, as CHCHD4 (human homologue of MIA40). However, neither its role as reductase, nor the molecular mechanisms underlying biogenesis of mitochondrial complexes are well understood. Additionally, when mitochondria receive an apoptotic stimulus AIF is translocated to the nucleus, where it binds other nuclear proteins to form the degradosome that will induce chromatinolysis. The interest in the design of new therapies to modulate caspase-independent apoptosis pathways has increased in the last years, making AIF a potential target to treat pathological disorders (cancer or degenerative diseases) in which this protein causes a defect or excess of apoptosis. The AIF redox state influences its conformation as well as a monomer-dimer equilibrium, and, by extension, its pro-apoptotic function by modulating the interaction with other proteins. Since one of the possibilities for modulating the AIF pro-apoptotic function means regulating its redox activity, it is therefore imperative to answer several questions related with such activity. Our research on human AIF focus on providing with some of these answers by understanding the molecular mechanisms linked to the AIF oxido-reduction processes and the consequences of the redox activity in the interaction with its partners in the different cellular components where it can be found.

 

Photosynthesis: a key energy transformation electron transfer chain dependent on flavoproteins

During photosynthesis an electron transfer chain produced through the formation of transitory short life complexes that involve the flavoenzyme ferredoxin-NADP+ reductase (FNR) convert light energy into chemical energy (as NADPH) usable by the cell. Under iron deficiency, some algae and cyanobacteria, like Anabaena, incorporate another flavoprotein to this chain, flavodoxin. Since the growth of such organisms is limited by the iron availability in many parts of the oceans, this chain supports a central role in global photosynthetic productivity. Despite the main biological function of this system is the production of reduction powder as NADPH, the reverse electron transfer processes also taking place in vivo.
Moreover, although the photosynthetic function was the first related to FNR, flavoproteins with FNR activity have been described in chloroplasts, figura-5-mmedinaphototropic and heterotrophic bacteria, apicoplasts and, animals and yeast mitochondria. Therefore, the electron flow in the photosynthetic and non-photosynthetic directions in Anabaena was chosen as a model for the study of the parameters involved in determining the catalytic mechanism and efficiency of enzymes of the FNR family. Our work has contributed to describe the structural features that determine key aspects of the interaction and electron transfer between the components of this system to efficiently perform their physiological functions. Simulation algorithms using QM/MM methods and experimental data have recently allowed producing a structural models of the catalytically competent binary complexes. Nevertheless, there are still numerous fundamental open questions about the regulation of the FNR function. Future research is required to reveal the main molecular intermediates and final species of the equilibrium mixture in the electron and hydride transfer processes within competent ternary complexes, and the contribution of proton transfer coupled to the efficiency of electron transfer in binary and ternary complexes. Altogether, the knowledge acquired in this system has allowed glimpsing the possibility to re-design functions of these flavoproteins and to produce heterologous enzymatic systems. Understanding how the protein environment modulates the flavin reduction potential has allowed producing a battery of variants with different redox properties.

 

Permanent collaborations in other flavoenzyme dependent systems.

NADPH-flavodoxin (ferredoxin) reductases (FPR) from bacteria: In collaboration with the groups of Dr. E. Ceccarelli and Dr. E. Orellano from the Universidad Nacional de Rosario, Argentina, we are comparatively studying bacterial and plastidic enzymes. Since in many bacteria these enzymes are either essential or involved in the response against oxidative stress, they are treated as interesting drug targets for the treatment of infections caused by pathogens.

Dehydrogenases and oxidases: In collaboration with Dr. Martinez’s, at the Centro de Investigaciones Biológicas, CSIC, Madrid, we have described the catalytic mechanism of the extracellular of the fungi flavoenzyme aryl-alcohol oxidase and identified key catalytic residues. This enzyme is involved in lignin degradation and biotechnological plant biomass utilization. Our structure-function studies have allowed describing at molecular detail its stereoselective mechanism in the oxidation of aromatic alcohols and aldehydes and to explore its great potential in the production of enantiomers and in synthesis of biopolymers. In collaboration with Dr. Nonato at the Universidade de São Paulo, Brasil, we are also characterizing different flavoenzymes with biomedical impact, such as the dihydroorotate dehidrogenase from Leshmania major and the L-amino acid oxidase from snake venom.

In addition we also maintain punctual collaborations with several other groups working with flavoenzymes for their structural and kinetic characterization.

Keywords: flavoenzyme biocatalysts • flavoenzyme mechanisms • fast kinetic methods • enzyme structures and dynamics • chemical biology

 

Methodologies used in our research:

  • Production of native and mutant proteins through the use of protein engineering techniques.
  • Homologous and heterologous protein expression in different microorganisms.
  • Purification of proteins (electrophoresis, chromatographic methods, HPLC, FPLC…).
  • Work under anaerobic conditions.
  • Absorption spectrometry: kinetic studies in steady-state, differential spectroscopy, midpoint reduction potential determination.
  • Use of transient kinetic techniques such as stopped-flow and laser flash photolysis.
  • Fluorescence spectroscopy and circular dichroism.
  • Protein crystallization and X-Ray diffraction for the determination of 3D protein structures.
  • Electron Paramagnetic Resonance related techniques (ESEEM, HYSCORE, ENDOR).
  • Isothermal Titration and Differential Scanning Calorimetries.
  • Docking, Molecular Dynamics and QM/MM simulations.

 

Relevant publications

1.- Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes. Laura Ledesma-García, Ana Sánchez-Azqueta, Milagros Medina*, Francisca Reyes-Ramírez* and Eduardo Santero. Scientific Reports 6:23848 (2016).

2.- Key residues regulating the reductase activity of the human mitochondrial apoptosis inducing factor. Raquel Villanueva, Carlos Marcuello, Alejandro Usón, M. Dolores Miramar, Maria Luisa Peleato, Ana Lostao, Santos A. Susin, Patricia Ferreira, and Milagros Medina. Biochemistry. 54, 5175-5184 (2015).

3.- A theoretical multiscale treatment of protein-protein electron transfer: the ferredoxin/ferredoxin-NADP+ reductase and flavodoxin/ferredoxin-NADP+ reductase systems. Suwipa Saen-oon, Israel Cabeza de Vaca, Milagros Medina, and Víctor Guallar. BBA-Bioenergetics. 184, 1530-1538 (2015).

4.- Dynamics of the active site architecture in plant-type Ferredoxin-NADP+ reductases catalytic complexes. Ana Sánchez-Azqueta, Daniela L. Catalano-Dupuy, Arleth López-Rivero, María Laura Tondo, Elena G. Orellano, Eduardo A. Ceccarelli, and Milagros Medina. BBA-Bioenergetics 1837, 1730-1738 (2014).

5.- Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic Apoptosis Inducing Factor. Patricia Ferreira, Raquel Villanueva, Marta Martínez-Júlvez, Beatriz Herguedas, Carlos Marcuello, Patricio Fernandez-Silva, Lauriane Cabon, Juan A. Hermoso, Anabel Lostao, Santos A. Susin and Milagros Medina. Biochemistry. 53, 4204-4215 (2014).

6.- ?

7.- Theoretical study of the mechanism of the hydride transfer between Ferredoxin NADP+ reductase and NADP+. The role of Tyrosine 303. Isaías Lans, Milagros Medina*, Edina Rosta*, Gerhard Hummer, Mireia García-Viloca, José M. Lluch, and Àngels González-Lafont. J. Am. Chem. Soc. 134, 20544-20553 (2012).

8.- Comparative genomics of Ceriporiopsis-subvermispora and Phanerochaete-chrysosporium provide insight into selective ligninolysis. Elena Fernández-Fueyo, Francisco J. Ruiz-Dueñas, Patricia Ferreira, +62 autores and Dan Cullen.  PNAS 109, 5458-5463 (2012).

9.- Role of key residues at the riboflavin kinase catalytic site of the bifunctional riboflavin kinase/FMN adenylyltransferase from Corynebacterium ammoniagenes. Ana Serrano, Susana Frago, Beatriz Herguedas, Marta Martínez-Júlvez, Adrián Velázquez-Campoy and Milagros Medina. Cellular Biochem. Biophys. 65, 57-68 (2013).

10.- Understanding the FMN cofactor chemistry within the Anabaena Flavodoxin environment. Isaías Lans, Susana Frago and Milagros Medina. BBA-Bioenergetics 1817, 2118-2127 (2012).

11.- Role of specific residues in charge transfer complex formation and hydride transfer between NADP+/H and Ferredoxin NADP+-reductase from Anabaena PCC 7119.José R. Peregrina, Ana Sánchez-Azqueta, Beatriz Herguedas, Marta Martínez-Júlvez and Milagros Medina. BBA-Bioenergetics 1797, 1638-1646 (2010).

12.- Evolutionary divergence of chloroplast FAD synthetase proteins. Inmaculada Yruela, Sonia Arilla, Milagros Medina and Bruno Contreras. BMC Evol. Biol. 10:311 (2010).

13.- Aryl-Alcohol Oxidase Involved in Lignin Degradation. A mechanistic study based on steady and pre-steady state kinetics and primary and solvent isotope effects with two alcohol substrates. Patricia Ferreira, Aitor Herández-Ortega, Beatriz Herguedas, Maria Jesús Martínez, Ángel T. Martínez and Milagros Medina. J. Biol. Chem.  284, 24840–24847 (2009).

14.- The puzzle of ligands binding to Corynebacterium ammoniagenes FAD synthetase. Susana Frago, Adrian Velázquez-Campoy and Milagros Medina. J. Biol. Chem. 284, 6610-6619 (2009).

15.- Hyperfine correlation spectroscopy and electron spin echo envelope modulation spectroscopy study of the two coexisting forms of the hemeprotein cytochrome c6 from Anabaena PCC 7119. Inés García Rubio, Pablo J. Alonso, Milagros Medina and Jesus. I. Martínez. Biophys. J. 96, 141-152 (2009).

 

Main research projects

1.- Flavoenzimas: mecanismos y dianas moleculares, patologías y aplicaciones biotecnológicas. BIO2016-75183-P. Ministerio de Economía y Competitividad (MINECO). Enero 2017-Diciembre 2019. Universidad de Zaragoza. Research Leader: Milagros Medina.

2.- Flavoenzyme dependent systems: from action mechanisms to biotechnological and sanitary applications. BIO2013-42978-P. Ministerio de Economía y Competitividad (MINECO). Enero 2014-Diciembre 2016. Universidad de Zaragoza. Research Leader: Milagros Medina.

3.- Seguimiento de los cambios conformacionales del dominio apoptótico del Factor de Inducción de Apoptosis Humano (hAIF) con marcaje selectivo de espín y espectroscopia de EPR. SGI (Universidad de Zaragoza) Enero 2015-Diciembre 2015. Research Leader: Patricia Ferreira.

4.- Retos enzimáticos, químicos y de ingeniería para la utilización de los recursos agroforestales no alimentarios (lignocelulosa) en una bio-economía más sostenible y menos contaminante. AC2014-00017-00-00. Ministerio de Economia y Competitividad (MINECO). Octubre 2014-Diciembre 2015. CIB-CSIC/Universidad de Zaragoza y otros. Research Leader: Susana Camarero Fernandez.

5.- Grupo Consolidado Biología Estructural (B18). Diputación General de Aragón 2014. Universidad de Zaragoza. Research Leader: Maria Luisa Peleato.

6.- Structure-based drug design for diagnosis and treatment of neurological diseases: dissecting and modulating complex function in the monoaminergic systems of the brain. COST ACTION CM1103. EU. 28 November  2011 Hasta: 27 November 2015. University of St. Andrews+18. Research Leader: Rona Ramsay.

7.- Mecanismos catalíticos en flavoenzimas: clave para su utilización biotecnológica o terapéutica. BIO2010-14983. Ministerio de Ciencia e Innovación. Universidad de Zaragoza. January 2011- September 2014. Research Leader: Milagros Medina.

8.- Propiedades proapoptótica y oxido-reductasa de la proteína mitocondrial AIF (factor de inducción de apoptosis): ¿Funciones biológicass independientes o complementarias?. Ref. 212363 (JIUZ-2012-BIO-01). SGI (Universidad de Zaragoza) Enero 2013-Diciembre 2013. Research Leader: Patricia Ferreira Neila.

9.- Flavoproteins and Flavoenzymes: Energy transformation and pharmacological targets. BIO2007-65890-C02-01. Dirección General de Investigación. Ministerio de Educación y Ciencia. Universidad de Zaragoza. October 2007-October 2010. Research Leader: Dr. Milagros Medina.

10.- Estudios Estructurales y Espectroscópicos mediante Difracción de Rayos X y EPR de Proteínas implicadas en Sistemas Biológicos de Transporte de Electrones. Vicerrectorado de Investigación-Apoyo A- Ibercaja.Universidad de Zaragoza. 2009. Research Leader: Dra. Marta Mª Martínez Júlvez.

 

Collaborators

Collaborations from BIFI

Dr. Juan Fernandez-Recio
Dr. Adrián Velázquez-Campoy
Dr. Inmaculada Yruela
Dr. Pier Paolo Bruscolini
Dr. Ramón Hurtado-Guerrero
Dr. José Alberto Carrodeguas
Dr. José Antonio Ainsa
Dr. Patricio Fernández

Collaborators from other Institutions

Instituto de Nanociencia de Aragón (INA)
– Dr. Ana Isabel Gracia Lostao
Universidad Nacional de Rosario, Rosario, Argentina
– Prof. Néstor Carrillo
– Dr. Néstor Cortez
– Dr. Eduardo Ceccarelli
– Dr. Elena Orellano
Centro de Investigaciones Biológicas, CSIC, Madrid
– Dr. Angel Martínez
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC. Sevilla
– Prof. Miguel A. de la Rosa
– Dr. Manuel Hervás
– Dr. José A. Navarro
Instituto Química Física-Rocasolano, CSIC, Madrid
– Dr. Juan A. Hermoso
IBB and Universidad Autónoma de Barcelona, Barcelona
– Dra. Mireia García-Viloca
– Dr. José M. Lluch
– Dra. Angels González
Barcelona Supercomputing Centre, Barcelona
– Dr. Victor Guallar
Universidad Pablo de Olavide, Sevilla
– Dra. Francisca Reyes
Centre de Recherche des Cordeliers, Paris, France 
– Dr. Santos Susín
Universita degli Studi di Bari
– Dr. Maria Barile
Universidad de Sao Paolo, Brasil
– Dr. M Cristina Nonato
Clark University, Biology Department, USA
– Dr. David Scott Hibbett
University of Oulu. Biocenter Oulu, and Faculty of Biochemistry and Molecular Medicine, Findland.
– Dr. André H. Juffer
Wageningen University, Laboratory of Biochemistry, Netherlands
– Prof. Willem van Berkel
University of Turku, Molecular Plant Biology, Finland
– Dr. Paula Mulo

Protein Misfolding
and Amyloid Aggregation

Head of the Research Line:

Dr. Nunilo Cremades

Researchers:

Dr. Nunilo Cremades, group leader
José Daniel Camino, PhD student
Marta Castellana, PhD student (shared with Prof. Chris Dobson, Univ. Cambridge, UK)
Pablo Gracia, PhD student

 

MAIN LINE OF RESEARCH

The phenomenon of protein misfolding and amyloid aggregation has emerged in recent years as a subject of fundamental importance in a wide range of scientific disciplines such as physics, chemistry, biology and medicine. This explosion of interest on the process of protein misfolding and amyloid aggregation has primarily resulted from the recognition that approximately 50 human diseases and disorders are associated with this process, some of them among the most common and debilitating medical conditions in the modern world, including Alzheimer’s, Parkinson’s and type II diabetes.

Despite the social and economical impact of some of these diseases, little is known about the molecular origins and mechanisms of protein amyloid aggregation and its associated toxicity. The research conducted in the group led by Dr. Nunilo Cremades aims to address these fundamental questions by combining a wide range of biophysical techniques, including state-of-the-art single-molecule fluorescence, with cell biology experiments.

fig1

Figure 1. The development and application of single-molecule fluorescence techniques have allowed us to investigate in unprecedented detail amyloid aggregation and to discover new possible therapeutic targets. Cremades N. et al. Cell 2012.

fig2

Figure 2. We have recently been able to purify highly stable amyloid oligomeric species of alpha-synuclein, the protein whose aggregation and deposition is linked with the development of Parkinson’s disease, and show that these species are highly cytotoxic and have general properties common to other amyloid oligomers. By combining a wide range of biophysical methods with cryo-EM image reconstruction techniques we were able to obtain three-dimensional structural models and reveal the quaternary structural architectures of toxic alpha-synuclein amyloid oligomers. Chen SW. et al. PNAS USA 2015.

 

OTHERS LINES OF RESEARCH

The group is also interested in understanding the role of intrinsic structural disorder in protein function and disease.

fig3

Figure 3. Typical phase diagram for a natively ordered and intrinsically disordered protein. We recently characterised the energy landscape of a naturally occurring intrinsically disordered enzyme. This protein acquires a wide range of molten globule-like, pre-molten globule-like and random coil-like conformations depending on the solution conditions. Zambelli and Cremades et al. Mol. Biosyst. 2012.

fig4

Figure 4. The presence of a specific type of structural disorder (molten globule-like conformations) correlates with the ability of human lysozyme to form amyloid fibrils (I56T mutant responsible for a hereditary form of systemic amyloidosis). Dhulesia and Cremades et al. J. Am. Chem. Soc. 2010

 

Relevant publications

1.- Calcium is a key factor in alpha-synuclein-induced neurotoxicity. Angelova P.R., Ludtmann M.H., Horrocks M.H., Negoda A., Cremades N., Klenerman D., Dobson C.M., Wood N.W., Pavlov E.V., Gandhi S. Abramov A.Y. J. Cell Sci. (2016) May 1;129(9):1792-801.

2.- Amyloid-β and α-synuclein decreases the level of metal-catalyzed reactive oxygen species by radical scavenging and redox silencing. Pedersen J.T., Chen S.W., Borg C.B., Ness S., Bahl J.M., Heegaard N.H., Dobson C.M., Hemmingsen L., Cremades N. (co-corresponding author) & Teilum K. J. Am. Chem. Soc. (2016) Mar 30;138(12):3966-9.

3.- Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading. Iljina M., Garcia G.A., Horrocks M.H., Tosatto L., Choi M.L., Ganzinger K.A., Abramov A.Y., Gandhi S., Wood N.W., Cremades N., Dobson C.M., Knowles T.P. & Klenerman D. Proc. Natl. Acad. Sci U.S.A. (2016) Mar 1; 113(9):E1206-15.

4.- Single-molecule imaging of individual amyloid protein aggregates in human biofluids. Horrocks M.H., Lee S.F., Gandhi S., Magdalinou N.K., Chen S.W., Devine M.J., Tosatto L., Kjaergaard M., Beckwith J.S., Zetterberg H., Iljina M., Cremades N., Dobson C.M., Wood N.W. & Klenerman D. ACS Chem. Neurosci. (2016) Mar 16;7(3):399-406.

5.- Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants. Tosatto L., Horrocks M.H., Dear A.J., Knowles T.P., Dalla Serra M., Cremades N., Dobson C.M. & Klenerman D. Sci. Rep. (2015) Nov 19;5:16696.

6.- Alpha-synuclein oligomers interact with metal ions to induce oxidative stress and neuronal death in Parkinson’s disease. Deas E., Cremades N., Angelova P.R., Ludtmann M.H., Yao Z., Chen S.W., Horrocks M.H., Banushi B., Little D., Devine M.J., Gissen P., Klenerman D., Dobson C.M., Wood N.W., Gandhi S. & Abramov AY. Antioxid. Redox Signal. (2016) Mar 1;24(7):376-91.

7.- Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of alpha-synuclein oligomers. Horrocks M.H., Tosatto L., Dear A.J., Garcia G.A., Iljina M., Cremades N., Dalla Serra M., Knowles T.P., Dobson C.M. & Klenerman D. Anal. Chem. (2015) Sep 1;87(17):8818-26.

8.- Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation. Chen S.W., Drakulic S., Deas E., Ouberai M., Aprile F.A., Arranz R., Ness S., Roodveldt C., Guilliams T., De-Genst E.J., Klenerman D., Wood N.W., Knowles T.P.J., Alfonso C., Rivas G., Abramov A.Y., Valpuesta J.M., Dobson C.M. & Cremades N.(corresponding author). Proc. Natl. Acad. Sci U.S.A. (2015) Apr 21;112(16):E1994-2003.

9.- Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson’s disease. Tóth G., Gardai S.J., Zago W., Bertoncini C.W., Cremades N., Roy S.L., Tambe M.A., Rochet J.C., Galvagnion C., Skibinski G., Finkbeiner S., Bova M., Regnstrom K., Chiou S.S., Johnston J., Callaway K., Anderson J.P., Jobling M.F., Buell A.K., Yednock T.A., Knowles T.P., Vendruscolo M., Christodoulou J., Dobson C.M., Schenk D. & McConlogue L. PLoS One (2014) Feb 14;9(2):e87133. doi: 10.1371/journal.pone.0087133.

10.- Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain. Aprile F.A., Dhulesia A., Stengel F., Roodveldt C., Benesch J.L., Tortora P., Robinson C.V., Salvatella X., Dobson C.M. & Cremades N. (corresponding author). PLoS One (2013) Jun 28;8(6):e67961. doi: 10.1371/journal.pone.0067961.

11.- Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cremades N., Cohen S.I., Deas E., Abramov A.Y., Chen A.Y., Orte A., Sandal M., Clarke R.W., Dunne P., Aprile F.A., Bertoncini C.W., Wood N.W., Knowles T.P.J., Dobson C.M. & Klenerman D. Cell (2012) 149(5): 1048-59.

12.- Insights in the (un)structural organization of Bacillus pasteurii UreG, an intrinsically disordered GTPase enzyme. Zambelli B. and Cremades N. (co-first author), Neyroz P., Turano P., Uversky V.N. & Ciurli S. Mol. Biosyst. (2012) 8(1): 220-8.

13.- Local cooperativity in an amyloidogenic state of human lysozyme observed at atomic resolution. Dhulesia A., Cremades N., Kumita J.R., Hsu S.T., Mossuto M.F., Dumoulin M., Nietlispach D., Akke M., Salvatella X. & Dobson C.M. J. Am. Chem. Soc. (2010) 132(44): 15580-8.

14.- On the mechanism of nonspecific inhibitors of protein aggregation: dissecting the interactions of alpha-synuclein with Congo red and Lacmoid. Lendel C., Bertoncini C.W., Cremades N., Waudby C.A., Vendruscolo M., Dobson C.M., Schenk D., Christodoulou J. & Toth G. Biochemistry (2009) 48(35): 8322-34.

15.- The native-state ensemble of proteins provides clues for folding, misfolding and function. Cremades N., Sancho J. & Freire E. Trends Biochem. Sci. (2006) 31(9): 494-6.

 

Main research projects

1.- Defining alpha-synuclein conformers responsable for Parkinson’s disease phenotypes in mice (ID 12708). Funding body: The Michael J. Fox Foundation (USA). PI: Dr. Laura Volpicelli-Daley and Dr. Nunilo Cremades. Start date: 1/05/2016, End date: 30/04/2018. Funds: 200,000$.

2.- Molecular basis and structural determinants of cellular toxicity of amyloid aggregation in Parkinson’s disease (BFU2015-64119-P). Funding body: Spanish Ministry of Economy and Competitiveness. PI: Dr. Nunilo Cremades. Start date: 1/01/2016, End date: 31/12/2018. Funds: 189,728 €.

3.- JIUZ-2015-BIO-03. Funding body: University of Zaragoza. PI: Dr. Nunilo Cremades. Start date: 1/01/2016, End date: 31/12/2016. Funds: 2,000 €.

4.- Single-molecule fluorescence equipment for the study of biomolecules (UNZA15-EE-2922). Funding body: Spanish Ministry of Economy and Competitiveness–FEDER. PI: Dr. Nunilo Cremades. Start date: 1/01/2017. Funds: 323,049.40 €.

 

Collaborators

  • Prof. Christopher M. Dobson (University of Cambridge, UK)
  • Dr. Laura Volpicelli-Daley (University of Alabama, USA)
  • Prof. Fabrizio Chiti (University of Florence, Italy)
  • Dr. Alfonso De Simone (Imperial College London, UK)
  • Prof. Felix Goñi (Basque Centre for Biophysics, Spain)
  • Prof. Paola Picotti (ETH Zurich, Switzerland)
  • Dr. Joseph Mazzulli (Northwestern University, USA)

 

Contact

Dr. Nunilo Cremades
Ramón y Cajal Researcher (Principal Investigator)
Institute for Biocomputation and Physics of Complex Systems
University of Zaragoza
Mariano Esquillor S/N. Edificio I+D+i
Zaragoza 50018, Spain
Phone: +34-876555417
Email:  ncc@unizar.es

Clinical Diagnosis
and Drug Delivery

Head of the Research Line:

Olga Abian Franco

Researchers:

Rafael Clavería, Predoctoral Student
María Arruebo, Predoctoral Student
Alberto Rodrigo, Predoctoral Student
Arturo Vinuesa, Predoctoral Student

 

SUMMARY OF THE RESEARCH LINE

Clinical Diagnosis

Differential Scanning Calorimetry (DSC) has recently emerged as a promising technique that provides useful information about serum/plasma interactomics (composition in proteins and metabolites, as well as their interactions). As a result of the illness, serum/plasma composition is altered and it is possible to discriminate between healthy individuals and patients with certain diseases.

A previous study performed in our group (25 healthy subjects and 60 gastric adenocarcinoma patients) showed that there were significant differences in the variables obtained from the calorimetric profiles of healthy and gastric adenocarcinoma patients and also between patients with different disease stages.

 

The validation and implementation of a methodology (DIGCAL) as a useful tool in diagnosing and monitoring relevant tumor pathologies (pancreatic ductal adenocarcinoma, preneoplasic pancreatic cystic lesions and stomach cancer), as well as in the isolation and identification of potential tumor biomarkers, are proposed in this project.

Calorimetric profiles of serum from healthy subjects and patients with the three types of cancer at different stages will be obtained before and after 6 months of clinical treatment. The multiparametric analysis of the thermal profiles will allow us to establish a clinical protocol for: 1/ screening a certain tumor process; 2/ classifying patients according to their tumour stage; 3/ monitoring the progression or control of the illness; 4/ identifying specific biomarkers for each disease type.
DIGCAL could reach the clinical level not only as a diagnosis tool, but also as a monitoring and tracking system of patients during the therapeutic treatment, adding value in prognostic or pharmacologic decisions. At the same time, identified potential biomarkers could be part of a user-friendly diagnosis kit for primary points of care.

Drug Delivery

Drug delivery is the method or process of administering a pharmaceutical compound to achieve a therapeutic effect in humans or animals. Drug delivery technologies modify drug release profile, absorption, distribution and elimination for the benefit of improving product efficacy and safety, as well as patient convenience and compliance. Drug release is from: diffusion, degradation, swelling, and affinity-based mechanisms. Most common routes of administration include the preferred non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes. Many medications such as peptide and protein, antibody, vaccine and gene based drugs, in general may not be delivered using these routes because they might be susceptible to enzymatic degradation or cannot be absorbed into the systemic circulation efficiently due to molecular size and charge issues to be therapeutically effective. For this reason many protein and peptide drugs have to be delivered by injection or a nanoneedle array. For example, many immunizations are based on the delivery of protein drugs and are often done by injection.

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Current efforts in the area of drug delivery include the development of targeted delivery in which the drug is only active in the target area of the body (for example, in cancerous tissues) and sustained release formulations in which the drug is released over a period of time in a controlled manner from a formulation. In order to achieve efficient targeted delivery, the designed system must avoid the host’s defense mechanisms and circulate to its intended site of action. Types of sustained release formulations include liposomes, drug loaded biodegradable microspheres and drug polymer conjugates. In this sense, Nanoparticles (NP) in Biomedicine represent a promising technology for drug transport and release. There are many possibilities for NP´s surface functionalization and so, many strategies for including drugs in them (allowing NP going mainly to their acting site) can be developed.

This research line represents a new strategy for including some antiviral compounds active against hepatitis C virus (HCV), that were previously developed in this group.

Several materials have been used:

1/ Cyclodextrins:

The chemical structure of CDs, cyclic oligosaccharides composed of α-1,4-glycosidic-linked glycosyl residues, provides them structural and physico-chemical properties that allow their use as molecular carriers.

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In their hydrophobic cavity a wide range of compounds ranging from ions to proteins can be trapped. In addition, CDs exhibit low toxicity and low immunogenicity, and they have been used in the pharmaceutical field by promoting CD-drug complexes in order to improve the absorption, distribution, metabolism, excretion, and toxicity (ADMET)-related properties of a drug (eg, solubility, stability, delivery and release, membrane permeability and absorption, toxicity). Currently, more than 30 products can be found in the market based on CD complexes.

2/ Shell Cross-Linked Polymeric Micelles

Cross-linked polymeric micelles (CLPM), formed by amphiphilic block copolymers, have been successful for biomedical applications. irst, they can fulfill those general requirements for drug delivery systems: water solubility, low toxicity, to increase the stability of the drug inside the living organisms, to facilitate cellular uptake compared to free drug, and to produce its controlled release at a specific location. Second, the amphiphilic nature of the constituent polymer results in a hydrophobic core and a hydrophilic shell that allows encapsulation of both types of drug. Third, these nanoparticles offer further stability under high dilution conditions, below the critical micellar concentration, compared to other polymeric micelles.

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Indeed, cross-linking avoids its disintegration in the bloodstream and the release of the drug before reaching the target cell. Particularly, fixation of the micelle structure by light-induced covalent cross-linking, mostly employing acrylate reactive groups, represents a clean and effective procedure to prepare stable polymer micelles that can hold either water-soluble and non-water soluble molecules and transport them through the bloodstream.

3/ Block Copolymers Micelles

Polymeric drug carriers are one of the current challenges of nanomedicine. Since the concept of physical drug encapsulation within polymeric aggregates was introduced, a significant number of polymer assemblies have been identified. In particular, the construction of amphiphilic block copolymer-based drug carriers is a subject of great interest and a stimulating topic of interdisciplinary research in chemistry, biology and materials science. In aqueous media, self-assembly of amphiphilic block copolymers (BCs) to minimize energetically unfavorable hydrophobic water interactions can lead to a variety of polymeric nanostructures including especially appealing spherical micelles and vesicles.

 

Relevant publications

1.- Polymeric micelles from block copolymers containing 2,6-diacylaminopyridine units for encapsulation of hydrophobic drugs.
Concellón A, Clavería-Gimeno E, Velázquez-Campoy A, Abian O, Piñol M, Oriol L.RSC Advances, 2016, 6, 24066–24075.

2.- Biophysical Screening for Identifying Pharmacological Chaperones and Inhibitors against Conformational and Infectious Diseases. Velazquez-Campoy A, Sancho J, Abian O, Vega S. Curr Drug Targets. 2016 Jan 31. [Epub ahead of print]

3.- Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena sp. PCC 7120. Botello-Morte L, Pellicer S, Sein-Echaluce VC, Contreras LM, Neira JL, Abian O, Velázquez-Campoy A, Peleato ML, Fillat MF, Bes MT. Antioxid Redox Signal. 2016, 24, 173-185.

4.- On the link between conformational changes, ligand binding and heat capacity. Vega S, Abian O, Velazquez-Campoy A. Biochim Biophys Acta. 2015 Oct 14. pii: S0304-4165(15)00274-3.

5.- Shell Cross-Linked Polymeric Micelles as Camptothecin Nanocarriers for anti-HCV Therapy. Jiménez-Pardo I, González-Pastor R, Lancelot A, Claveria-Gimeno R, Velázquez-Campoy A, Abian O, Ros MB, and Sierra T. Macromol. Biosci. 2015, 15, 1381–1391.

6.- Su1990 A New Technology for the Classification of Patients With Gastric Adenocarcinoma Based on Differential Scanning Calorimetry Serum Thermograms. Vega S, Garcia-Gonzalez MA, Lanas A, Velazquez-Campoy A, Abian O.*. Gastroenterology 148(4): S-569 · April 2015

7.- Rescuing compound bioactivity in a secondary cell-based screening by using γ-cyclodextrin as a molecular carrier. Clavería-Gimeno R, Vega S, Grazu V, De la Fuente JM, Lanas A, Velazquez-Campoy A, Abian O.*. International Journal of Nanomedicine 2015, 10: 2249-2259.

8.- Deconvolution Analysis for Classifying Gastric Adenocarcinoma Patients Based on Differential Scanning Calorimetry Serum Thermograms. Vega S, Garcia-Gonzalez MA, Lanas A, Velazquez-Campoy A, Abian O.*. Scientific reports, 5:7988 (2015).

9.- Ionic liquids in water: a green and simple approach to improve activity and selectivity of lipases.
Filice M, Romero O,  Abian O, De las Rivas B and Palomo J.M. RSC Advances 4: 49115- 49122 (2014).

10.- A unified framework based on the binding polynomial for characterizing biological systems by isothermal titration calorimetry. Vega S., Abian O.*and Velazquez-Campoy A. Methods. 2014 pp: S1046-2023(14) 00316-8.

11.- Allosteric Inhibitors of the NS3 Protease From the Hepatitis C Virus. Abian O.*, Vega S., Sancho J., Velazquez-Campoy A. PLOSone 2013, 8 (7): 69773.

12.- NS3 protease from hepatitis C virus: Biophysical studies on an intrinsically disordered protein domain. Vega S., Neira J.L., Marcuello C.,  Lostao A., Abian O. * and Velazquez-Campoy A. International Journal of Molecular Sciences 2013, 14: 13282-13306.

13.- Experimental Validation of In Silico Target Predictions on Synergistic Protein Targets. Cortes-Ciriano I, Koutsoukas A, Abian O, Velazquez-Campoy A and Bender A. MedChemComm 2013, 4, 278–288.

14.- Altering the interfacial activation mechanism of a lipase by solid-phase selective chemical modification. López-Gallego F, Abian O, Guisán JM. Biochemistry. 2012 Sep 4;51(35):7028-36.

15.- Semisynthetic peptide-lipase conjugates for improved biotransformations. Romero O, Filice M, de las Rivas B, Carrasco-Lopez C, Klett J, Morreale A, Hermoso JA, Guisan JM, Abian O, Palomo JM. Chem Commun (Camb). 2012 Sep 18;48(72):9053-5.

 

Main research projects

Ongoing Projects:

1.- Analysis of protein/metabolites interactions in plasma serum using calorimetry: application as a quick and noninvasive diagnostic method for early detection and monitoring of tumoral digestive diseases (DIGCAL). Funding Institution: Health Institute Carlos III. From: January 2016 To: December 2018. Principal Investigator (PI): Olga Abian Franco.

2.- Validación de un nuevo método diagnóstico en suero, rápido no invasivo para detección precoz de cáncer de páncreas (PANCal). Funding Institution: Asociación Española de Gastroenterología (AEG). From: 2015 To: 2016. Principal Investigator (PI): Olga Abian Franco.

Former Projects:

3.- Adapted Nanoparticles for transport and specific release of drugs against hepatitis C virus (VHC).
Funding Institution: Health Institute Carlos III. From: 2011 To: 2013. Principal Investigator (PI): Olga Abian Franco.

4.- Implementation of in vitro e in vivo studies of anti-infectious compounds effective against Helicobacter pylori y el HCV (hepatitis C virus). Funding Institution: Health Institute Carlos III. From: 2008 To: 2011. Principal Investigator (PI): Olga Abian Franco.

 

Collaborators

Collaborations from BIFI

Dr. Adrián Velázquez-Campoy
Prof. Javier Sancho
Dr. Jose Luis Neira

Collaborators from other Institutions

James Graham Brown Cancer Center, University of Louisville, Louisville, KY, EEUU
PhD. Nichola Garbett

Instituto Aragonés de Ciencias de la Salud (I+CS), Zaragoza
Prof. Angel Lanas
Dra. Trinidad Serrano
Dra. Estela Solanas

Instituto de Ciencia de Materiales de Aragón (ICMA). Química Orgánica. Facultad de Ciencias.
Dra. Teresa Sierra
Prof. Luis Oriol
Prof. Milagros Piñol

Instituto de Nanociencia (INA), Universidad de Zaragoza, Zaragoza
Dr. Jesús Martínez de Lafuente
Dra. Valeria Grazú
Dra. Berta Saez

Universidad de San Jorge (USJ)
Prof. Victor López
Prof. Elisa Langa

Instituto de Catálisis y Petroleoquímica, CSIC, Madrid.
Dr. Jose Miguel Palomo
Dr. Fernando López Gallego
Dr. Jose Manuel Guisan

Universidad de Zaragoza
Dr. Jose Antonio Ainsa

Unidad de Investigación Traslacional, Hospital Universitario Miguel Servet, Zaragoza
Dra. Pilar Alfonso
Dra. Pilar Giraldo
Dr. Miguel Pocovi

Servicio de Microbiología-INIBIC. Complejo Hospitalario Universitario A Coruña, La Coruña
Dr. Francisco José Pérez-Llarena

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